1、therefore adv.因此, 所以=for that reason=consequently常用於連接兩個並列分句,其前加“and”或分號“;”。
He was ill, and therefore could not come. 他病了, 所以未能來。
He has broken his leg and therefore he can't walk.他摔壞了腿,因此不能走路了。
We do not have enough money. Therefore we cannot afford to buy the new car.我們的錢不夠,因此買不成這輛新車。
2、so conj. adv因而, 所以, 結果是
It was late, so we went home. 天晚了, 所以我們就回家去了。
He was sick, so they were quiet.他病了,所以他們很安靜。
3、hence adv.因此, 從此
The town was built among the hills, hence the name Hilltown. 該城鎮修建在群山之中,故取名'山城'。
It is very late; hence you must go to bed.時間已經很晚了,因此你必須睡覺去。(記住前面是分號,而不是逗號)
4、then adv因此,就
Go into the cave, then they won't see you. 躲進洞裡去,那他們就看不到你了。
5、accordingly adv.因此, 從而;所以,因此結果;因此(偶作連詞使用conj.)
He was asked to leave the city and accordingly he went.有人叫他離開該城市,所以他就走了。
You may arrange accordingly.你可以權宜處理。
Mr Foster has never been to China. Consequently / Hence he knows very little about it.福斯特先生從未去過中國,所以對中國了解得很少。
My car was broken down and consequently I was late. = in consequence
6、Thus adv.如此,像這樣;因此;於是
He sold his farm and thus he had enough money for his journey.他賣掉了農場,這樣他就有足夠的錢旅行了。
There has been no rain — thus, the crops are drying.天沒下雨,因此莊稼要枯死了。
2011年12月15日 星期四
2011年10月9日 星期日
p53 腫瘤抑制基因
p53為腫瘤抑制蛋白(也稱為p53蛋白或p53腫瘤蛋白),屬於最早發現的腫瘤抑制基因(或抑癌基因)之一。 p53蛋白能調節細胞週期和避免細胞癌變發生。 因此,p53蛋白被稱為基因組守護者。 總而言之,其角色為保持基因組的穩定性,避免突變發生。
功能
p53蛋白在避免癌症發生機制上扮演重要的角色,例如,細胞凋亡 (apoptosis) 、基因組穩定性 (genetic stability) 、抑制血管新生 (angiogenesis)。 p53蛋白通過下列之機構達成避免癌症發生:
當DNA受損時,p53蛋白能活化DNA修復蛋白 (DNA repair proteins)。
p53蛋白能抑制細胞生長週期停留於G1/S的節律點上,以達成DNA損壞辨識。
(若能將細胞於此節律點上停留夠久,DNA修護蛋白將有更充裕的時間修復DNA損壞部位,並繼續細胞的生長週期。)
若細胞的DNA受損已不能修復,p53蛋白能起始細胞凋亡程序,避免擁有不正常遺傳資訊的細胞繼續分裂生長。
活化的p53蛋白能接合於DNA,促使多個基因表現,包括基因WAF1/CIP1,其為p21蛋白之編碼基因。
p21接合於[[G1-S/CDK (CDK2)]] 和S/CDK複合體 (此蛋白在G1/S細胞週期節律點上有重要功能) 以抑制該複合體的活性。
當p21蛋白與CDK2形成複合體時,細胞將無法進入到細胞分裂的階段。(p21扮演細胞分裂剎車的角色)
而突變後的p53蛋白將可能喪失與DNA形成有效結合的能力,造成p21蛋白將無法形成,細胞分裂就會持續進行,無法停止。 因此,受損細胞將不受控制的進行細胞分裂,最終形成腫瘤。 根據最近的研究,p53蛋白與RB1程序經由p14ARF蛋白相互調節的可能性更加提高。
調節
p53蛋白藉由許多不同的壓力形式而激發其活性,其中包括但不僅僅侷限於DNA損傷 (包括 UV, IR或化學物質如過氧化氫 (hydrogen peroxide)所造成的損傷),氧化壓力 (oxidative stress),滲透壓力 (osmotic stress),核糖核苷酸缺乏 (nucleotide depletion) 和喪失調節癌基因表現能力。這些活性激發可由兩個主要的事件得出。首先,在受到壓力的細胞中,p53蛋白的半衰期 (half-life) 會突然的增加,造成p53蛋白在細胞中的累積。再來則是構型變化 (conformational change) 使得p53蛋白被激發成為轉錄調節因子 (transcription regulator)。
功能
p53蛋白在避免癌症發生機制上扮演重要的角色,例如,細胞凋亡 (apoptosis) 、基因組穩定性 (genetic stability) 、抑制血管新生 (angiogenesis)。 p53蛋白通過下列之機構達成避免癌症發生:
當DNA受損時,p53蛋白能活化DNA修復蛋白 (DNA repair proteins)。
p53蛋白能抑制細胞生長週期停留於G1/S的節律點上,以達成DNA損壞辨識。
(若能將細胞於此節律點上停留夠久,DNA修護蛋白將有更充裕的時間修復DNA損壞部位,並繼續細胞的生長週期。)
若細胞的DNA受損已不能修復,p53蛋白能起始細胞凋亡程序,避免擁有不正常遺傳資訊的細胞繼續分裂生長。
活化的p53蛋白能接合於DNA,促使多個基因表現,包括基因WAF1/CIP1,其為p21蛋白之編碼基因。
p21接合於[[G1-S/CDK (CDK2)]] 和S/CDK複合體 (此蛋白在G1/S細胞週期節律點上有重要功能) 以抑制該複合體的活性。
當p21蛋白與CDK2形成複合體時,細胞將無法進入到細胞分裂的階段。(p21扮演細胞分裂剎車的角色)
而突變後的p53蛋白將可能喪失與DNA形成有效結合的能力,造成p21蛋白將無法形成,細胞分裂就會持續進行,無法停止。 因此,受損細胞將不受控制的進行細胞分裂,最終形成腫瘤。 根據最近的研究,p53蛋白與RB1程序經由p14ARF蛋白相互調節的可能性更加提高。
調節
p53蛋白藉由許多不同的壓力形式而激發其活性,其中包括但不僅僅侷限於DNA損傷 (包括 UV, IR或化學物質如過氧化氫 (hydrogen peroxide)所造成的損傷),氧化壓力 (oxidative stress),滲透壓力 (osmotic stress),核糖核苷酸缺乏 (nucleotide depletion) 和喪失調節癌基因表現能力。這些活性激發可由兩個主要的事件得出。首先,在受到壓力的細胞中,p53蛋白的半衰期 (half-life) 會突然的增加,造成p53蛋白在細胞中的累積。再來則是構型變化 (conformational change) 使得p53蛋白被激發成為轉錄調節因子 (transcription regulator)。
2011年9月29日 星期四
10x PBS
To make 10X PBS Buffer, dissolve the following reagents in 800 ml ultrapure water.
NaCl 80 g
KCl 2 g
Na2HPO4 14.4 g
KH2PO4 2.4 g
Adjust pH of PBS Buffer Solution to 7.4 with HCl.
Bring volume to 1 liter, autoclave or sterilize by filtration.
Molecular Cloning
1X PBS (10 mM)
8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
in 800 ml of distilled H2O.
Adjust the pH to 7.4 with HCl. Add H2O to 1 liter. Sterilize by autoclave.
10X PBS
Molarity of 1X PBS is 10mM. To prepare 20mM PBS take 16g of NaCl, 0.4 g KCl, 2.88 g Na2HPO4 0.48 g KH2PO4 in 800 ml of distilled H2O. Adjust the pH to 7.4 with HCl. Add H2O to 1 liter.
NaCl 80 g
KCl 2 g
Na2HPO4 14.4 g
KH2PO4 2.4 g
Adjust pH of PBS Buffer Solution to 7.4 with HCl.
Bring volume to 1 liter, autoclave or sterilize by filtration.
Molecular Cloning
1X PBS (10 mM)
8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
in 800 ml of distilled H2O.
Adjust the pH to 7.4 with HCl. Add H2O to 1 liter. Sterilize by autoclave.
10X PBS
- Dissolve the following in 800ml distilled H2O.
- 80g of NaCl
- 2.0g of KCl
- 14.4g of Na2HPO4
- 2.4g of KH2PO4
- Adjust pH to 7.4.
- Adjust volume to 1L with additional distilled H2O.
- Sterilize by autoclaving.
Molarity of 1X PBS is 10mM. To prepare 20mM PBS take 16g of NaCl, 0.4 g KCl, 2.88 g Na2HPO4 0.48 g KH2PO4 in 800 ml of distilled H2O. Adjust the pH to 7.4 with HCl. Add H2O to 1 liter.
2011年9月22日 星期四
流式細胞儀Flow cyotmetry
使用流式細胞儀注意事項
1. 調整完條件後,每一個試驗組可以先試跑條件,確定都在相同的區域下,再開始收數據
2. 細胞經由Alcohol固定之後,會變得很黏,所以要直接先用冰酒精(4度C)打散,以免之後黏成一團,無法進行實驗
3.
1. 調整完條件後,每一個試驗組可以先試跑條件,確定都在相同的區域下,再開始收數據
2. 細胞經由Alcohol固定之後,會變得很黏,所以要直接先用冰酒精(4度C)打散,以免之後黏成一團,無法進行實驗
3.
2011年9月21日 星期三
CCAAT-enhancer-binding proteins
CCAAT-enhancer-binding proteins (or C/EBPs) are a family of transcription factors, composed of six members called C/EBP α to C/EBP ζ. They promote the expression of certain genes through interaction with their promoter. Once bound to DNA, C/EBPs can recruit so-called coactivators (such as CBP, see ref. 2) that, in turn, can open up chromatin structure, or recruit basal transcription factors.
目前
C/EBP proteins interact with the CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine) box motif, which is present in several gene promoters. They are characterized by a highly conserved basic-leucine zipper (bZIP) domain at the C-terminus. This domain is involved in dimerization and DNA binding, like other transcription factors of the leucine zipper family like c-Fos and Jun. C/EBPs bZIP domain structure is composed of an α-helix that forms a coiled coil structure when it dimerizes. The different members of C/EBP family can form homodimers, heterodimers with another form of the C/EBPs and with other transcription factors that may or may not contain the leucine zipper domain. The dimerization is required for the activity of C/EBPs to bind specifically to DNA through a palindromic sequence in the major groove of the DNA. The C/EBP proteins also contain activation domains at the N-terminus and regulatory domains.
These proteins are found in hepatocytes, adipocytes, hematopoietic cells, spleen, kidney, brain and many others organs. C/EBPs proteins are involved in different cellular responses like in the control of cellular proliferation, growth and differentiation, metabolism, immunology and many others. All the members of the C/EBP family, except C/EBPγ that lacks transcriptional activation domain, can induce transcription, through their activation domains, by interacting with components of the basal transcription apparatus. Their expression is regulated at multiple levels through hormones, mitogens, cytokines, nutrients, etc.
The C/EBPα, -β, -γ and -δ genes are intronless and C/EBPε and -ζ have respectively two and four exons that lead in the case of C/EBP ε to four isoforms due to an alternative use of promoters and splicing. For C/EBPα and -β, different sizes of polypeptides can be produced by alternative use of initiation codons due to weak ribosome scanning mechanisms. The mRNA of C/EBPα can lead to two polypeptides and for C/EBPβ three different polypeptides are made: LAP* (38 kDa), LAP (35 kDa) and LIP (20 kDa). The most translated isoform is LAP, then LAP* and LIP; the latter can act as an inhibitor of the other C/EBPs by forming non-functional heterodimers.
This protein is expressed in the mammalian nervous system and has many implications in the nerve cells. C/EBPβ plays a role in neuronal differentiation, in learning and memory process, glial or neuronal cell functions and neurotrophic factory expression.
C/EBPβ function is regulated via multiple mechanisms: phosphorylation; acetylation; activation and repression via other transcription factors, oncogenic elements or chemokines; autoregulation, etc. C/EBPβ can interact with different proteins, such as CREB, NF-κB and others, leading to a trans-activation potential. Phosphorylation of C/EBPβ can have an activation or a repression effect. For example, phosphorylation of threonine 235 in human C/EBPβ, or of threonine 188 in mouse and rat C/EBPβ, is important for C/EBPβ trans-activation capacity; phosphorylation(s) of C/EBPβ in its regulatory domain can also modulate its function.
目前
C/EBP proteins interact with the CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine) box motif, which is present in several gene promoters. They are characterized by a highly conserved basic-leucine zipper (bZIP) domain at the C-terminus. This domain is involved in dimerization and DNA binding, like other transcription factors of the leucine zipper family like c-Fos and Jun. C/EBPs bZIP domain structure is composed of an α-helix that forms a coiled coil structure when it dimerizes. The different members of C/EBP family can form homodimers, heterodimers with another form of the C/EBPs and with other transcription factors that may or may not contain the leucine zipper domain. The dimerization is required for the activity of C/EBPs to bind specifically to DNA through a palindromic sequence in the major groove of the DNA. The C/EBP proteins also contain activation domains at the N-terminus and regulatory domains.
These proteins are found in hepatocytes, adipocytes, hematopoietic cells, spleen, kidney, brain and many others organs. C/EBPs proteins are involved in different cellular responses like in the control of cellular proliferation, growth and differentiation, metabolism, immunology and many others. All the members of the C/EBP family, except C/EBPγ that lacks transcriptional activation domain, can induce transcription, through their activation domains, by interacting with components of the basal transcription apparatus. Their expression is regulated at multiple levels through hormones, mitogens, cytokines, nutrients, etc.
The C/EBPα, -β, -γ and -δ genes are intronless and C/EBPε and -ζ have respectively two and four exons that lead in the case of C/EBP ε to four isoforms due to an alternative use of promoters and splicing. For C/EBPα and -β, different sizes of polypeptides can be produced by alternative use of initiation codons due to weak ribosome scanning mechanisms. The mRNA of C/EBPα can lead to two polypeptides and for C/EBPβ three different polypeptides are made: LAP* (38 kDa), LAP (35 kDa) and LIP (20 kDa). The most translated isoform is LAP, then LAP* and LIP; the latter can act as an inhibitor of the other C/EBPs by forming non-functional heterodimers.
This protein is expressed in the mammalian nervous system and has many implications in the nerve cells. C/EBPβ plays a role in neuronal differentiation, in learning and memory process, glial or neuronal cell functions and neurotrophic factory expression.
C/EBPβ function is regulated via multiple mechanisms: phosphorylation; acetylation; activation and repression via other transcription factors, oncogenic elements or chemokines; autoregulation, etc. C/EBPβ can interact with different proteins, such as CREB, NF-κB and others, leading to a trans-activation potential. Phosphorylation of C/EBPβ can have an activation or a repression effect. For example, phosphorylation of threonine 235 in human C/EBPβ, or of threonine 188 in mouse and rat C/EBPβ, is important for C/EBPβ trans-activation capacity; phosphorylation(s) of C/EBPβ in its regulatory domain can also modulate its function.
2011年9月17日 星期六
巨噬細胞的胰島素訊息傳遞
The Macrophage at the crossroads of insulin resistance and atherosclerosis
Circulation Research 2007 100:1546-1555
巨噬細胞在胰島素抗性與動脈粥狀硬化上扮演很重要的角色
巨噬細胞呈現胰島素抗性時,對於ER stress會更為敏感,容易apoptosis
尤其是營養缺乏(被剝奪)(nutrient deprivation) 膽固醇loading 氧化的LDL過多的情形
而已經形成胰島素抗性的巨噬細胞會更容易死亡也造成清除機制受損 將會造成 動脈粥狀硬化的形成
insulin signaling
巨噬細胞內擁有大部分的胰島素傳遞路徑 除了IRS1 以及 GLUT4 (因此 胰島素並不會促進葡萄糖吸入巨噬細胞, GLUT1)
胰島素對於巨噬細胞的調控目前仍不清楚 大致上可以增加存活率、蛋白合成與分泌、Phagocytosis以及先天免疫(innate immunity)
Circulation Research 2007 100:1546-1555
巨噬細胞在胰島素抗性與動脈粥狀硬化上扮演很重要的角色
巨噬細胞呈現胰島素抗性時,對於ER stress會更為敏感,容易apoptosis
尤其是營養缺乏(被剝奪)(nutrient deprivation) 膽固醇loading 氧化的LDL過多的情形
而已經形成胰島素抗性的巨噬細胞會更容易死亡也造成清除機制受損 將會造成 動脈粥狀硬化的形成
insulin signaling
巨噬細胞內擁有大部分的胰島素傳遞路徑 除了IRS1 以及 GLUT4 (因此 胰島素並不會促進葡萄糖吸入巨噬細胞, GLUT1)
胰島素對於巨噬細胞的調控目前仍不清楚 大致上可以增加存活率、蛋白合成與分泌、Phagocytosis以及先天免疫(innate immunity)
2011年8月7日 星期日
十種電子郵件表達"感謝"之意的方式
以下資訊來源為englishtown
http://www.englishtown.com/community/channels/article.aspx?articlename=196-thankyou
Top ten ways to say “Thank you” in an English email/
十種電子郵件表達"感謝"之意的方式
不論你正寫一封電子郵件給你的客戶、你的經理或是你的同事,感謝的話語永遠都不夠的。大家都想得到別人的感謝,對於在過去及未來的日子給予你協助的這些閱讀者,我們要表達感謝之意!
Whether you’re writing to a client, to a manager or to a colleague, you can’t thank people enough in your emails. Everyone wants to feel appreciated, so thank your readers for what they have already done for you, and thank them for what you want them to do in the future! You’ll find that some kind words combined with sincere appreciation of their efforts will go a long way.
================================
1. At the beginning of the email
最有效來表達感謝之意就是開門見山。如果在未來的日子你仍然需要獲得他人的協助,在信件中適當的語氣能讓閱讀者感受到被感謝是相當重要的。
Thanking your reader is a wonderful way of opening an email. It sets the right tone and makes the reader feel appreciated, which is particularly vital if you require future help from them.
2. Thank you for contacting us.
如果某人詢問關於你的公司服務項目,可以在信的開頭使用這個句子,來表達感謝他們對公司感興趣的意願。
If someone writes to enquire about your company’s services, begin your email with this sentence. Show your appreciation for their interest in working with your company.
3. Thank you for your prompt reply.
當客戶或同事在很短的時間回覆上次來信,你可以這樣來表達感謝之意。如果不是在很短的時間回覆的話,你就不需要加上“prompt”或也可以這樣表示「Thank you for getting back to me.」
When a client or colleague replies to a previous email in a short amount of time, notice and acknowledge this. If the reply wasn’t quick, simply removing “prompt” will work, or, you can opt for, “Thank you for getting back to me.”
4. Thank you for providing the requested information.
詢問某人問題,並且他們利用一些時間來回覆給你,你可以利用這個句子來表達非常感激他們所做的一切。
If you have asked someone for information, and they took the time to send it to you, use this sentence to demonstrate that you value what they’ve done.
5. Thank you for all your assistance.
如果某人以他們的方式給予協助,你就可以感謝他們!如果你想要更明確表達感謝他們所做的一切,您可以使用這個句子:「I truly appreciate ... your help in resolving the problem.」
If someone has gone out of their way to help you, thank them! If you want to offer more specific recognition for what they have done, follow this sentence with, “I truly appreciate … your help in resolving the problem.”
6. Thank you raising your concerns.
即使你的客戶或經理人已寫下他們表達對於你在工作上的關心之意,你仍然可以感謝他們。表示你非常重視他們。或者,你也可以這樣寫:「Thank you for your feedback.」
Even if a client or manager writes to express some concerns they have regarding your work, you can still thank them. This shows that you value their input and will take their concerns seriously. Alternatively, you may wish to use, “Thank you for your feedback.”
7. At the end of the email
閱讀者通常會在信的開頭表達感謝之意,如果在信的結尾重複表達感謝之意,代表你希望在未來能獲得對方的協助,並且能獲得正面回應。
While thank yous at the beginning of an email are typically written to thank the reader for past actions, thank yous at the end of an email tend to imply you are thanking the reader for a future action. By showing your appreciation in advance, you are more likely to get a positive reaction.
8. Thank you for your kind cooperation.
如果你需要與閱讀者的合作協助某事,那就先表達感謝他們的合作。
If you need the reader to cooperate by assisting you with something, then thank them in advance for their cooperation.
9. Thank you for your attention to this matter.
與上面一個句子相同,這個句子暗示閱讀者的任何協助將獲得感激。
Similar to above, this sentence implies that you would appreciate the readers’ further assistance.
10. Thank you for your understanding.
如果所做一切將使閱讀者感到不便或負面的影響,可以使用這個句子。
Use this sentence if you’ve shared something that may inconvenience or negatively impact the reader.
11. Thank you for your consideration.
如果你請求一個好處或機會,譬如申請一個新的工作,你可以在結尾使用這個句子。
If you are requesting a benefit or an opportunity, such as when you apply for a new job, end your email with this sentence.
12. Thank you again for everything you've done.
這個句子通常放在句末,與前面幾個句子在使用上有些不同。如果你在信的開頭已表達感謝之意,你就可以使用這個句子,你希望能再次感謝他們過去付出的所有熱忱。
This sentence, which is used at the end, is a bit different from those above. Use this if you have already thanked the reader at the beginning of the email, but due to their great efforts, you wish to thank them again for their past actions.
http://www.englishtown.com/community/channels/article.aspx?articlename=196-thankyou
Top ten ways to say “Thank you” in an English email/
十種電子郵件表達"感謝"之意的方式
不論你正寫一封電子郵件給你的客戶、你的經理或是你的同事,感謝的話語永遠都不夠的。大家都想得到別人的感謝,對於在過去及未來的日子給予你協助的這些閱讀者,我們要表達感謝之意!
Whether you’re writing to a client, to a manager or to a colleague, you can’t thank people enough in your emails. Everyone wants to feel appreciated, so thank your readers for what they have already done for you, and thank them for what you want them to do in the future! You’ll find that some kind words combined with sincere appreciation of their efforts will go a long way.
================================
1. At the beginning of the email
最有效來表達感謝之意就是開門見山。如果在未來的日子你仍然需要獲得他人的協助,在信件中適當的語氣能讓閱讀者感受到被感謝是相當重要的。
Thanking your reader is a wonderful way of opening an email. It sets the right tone and makes the reader feel appreciated, which is particularly vital if you require future help from them.
2. Thank you for contacting us.
如果某人詢問關於你的公司服務項目,可以在信的開頭使用這個句子,來表達感謝他們對公司感興趣的意願。
If someone writes to enquire about your company’s services, begin your email with this sentence. Show your appreciation for their interest in working with your company.
3. Thank you for your prompt reply.
當客戶或同事在很短的時間回覆上次來信,你可以這樣來表達感謝之意。如果不是在很短的時間回覆的話,你就不需要加上“prompt”或也可以這樣表示「Thank you for getting back to me.」
When a client or colleague replies to a previous email in a short amount of time, notice and acknowledge this. If the reply wasn’t quick, simply removing “prompt” will work, or, you can opt for, “Thank you for getting back to me.”
4. Thank you for providing the requested information.
詢問某人問題,並且他們利用一些時間來回覆給你,你可以利用這個句子來表達非常感激他們所做的一切。
If you have asked someone for information, and they took the time to send it to you, use this sentence to demonstrate that you value what they’ve done.
5. Thank you for all your assistance.
如果某人以他們的方式給予協助,你就可以感謝他們!如果你想要更明確表達感謝他們所做的一切,您可以使用這個句子:「I truly appreciate ... your help in resolving the problem.」
If someone has gone out of their way to help you, thank them! If you want to offer more specific recognition for what they have done, follow this sentence with, “I truly appreciate … your help in resolving the problem.”
6. Thank you raising your concerns.
即使你的客戶或經理人已寫下他們表達對於你在工作上的關心之意,你仍然可以感謝他們。表示你非常重視他們。或者,你也可以這樣寫:「Thank you for your feedback.」
Even if a client or manager writes to express some concerns they have regarding your work, you can still thank them. This shows that you value their input and will take their concerns seriously. Alternatively, you may wish to use, “Thank you for your feedback.”
7. At the end of the email
閱讀者通常會在信的開頭表達感謝之意,如果在信的結尾重複表達感謝之意,代表你希望在未來能獲得對方的協助,並且能獲得正面回應。
While thank yous at the beginning of an email are typically written to thank the reader for past actions, thank yous at the end of an email tend to imply you are thanking the reader for a future action. By showing your appreciation in advance, you are more likely to get a positive reaction.
8. Thank you for your kind cooperation.
如果你需要與閱讀者的合作協助某事,那就先表達感謝他們的合作。
If you need the reader to cooperate by assisting you with something, then thank them in advance for their cooperation.
9. Thank you for your attention to this matter.
與上面一個句子相同,這個句子暗示閱讀者的任何協助將獲得感激。
Similar to above, this sentence implies that you would appreciate the readers’ further assistance.
10. Thank you for your understanding.
如果所做一切將使閱讀者感到不便或負面的影響,可以使用這個句子。
Use this sentence if you’ve shared something that may inconvenience or negatively impact the reader.
11. Thank you for your consideration.
如果你請求一個好處或機會,譬如申請一個新的工作,你可以在結尾使用這個句子。
If you are requesting a benefit or an opportunity, such as when you apply for a new job, end your email with this sentence.
12. Thank you again for everything you've done.
這個句子通常放在句末,與前面幾個句子在使用上有些不同。如果你在信的開頭已表達感謝之意,你就可以使用這個句子,你希望能再次感謝他們過去付出的所有熱忱。
This sentence, which is used at the end, is a bit different from those above. Use this if you have already thanked the reader at the beginning of the email, but due to their great efforts, you wish to thank them again for their past actions.
2011年8月1日 星期一
2011年7月26日 星期二
[自我成長]二十年都是人才
5大關鍵,讓你二十年後依然是人才
2006-06 天下雜誌 349期
你是正在力爭上游的基層員工、公司的當紅炸子雞、中高階主管、還是即將退休的資深元老?
無論你身在哪一個階段,當職場趨勢已從企業端的「終身雇用」轉變為個人端的「終身就業」,你必須時時增加自己的競爭力,即使二十年後,依然是企業搶著要的人才。
綜合日本趨勢專家大前研一、奇異前任執行長威爾許的觀點,以及《日經商業週刊》的報導,以下五件事,是你為自己加分的關鍵思考:
1.不管坐什麼位置,都要保持學習的習慣
出社會工作十年到十五年左右,會有一種「上下卡住」的閉塞感與無力感。因為,這個階段的上班族雖然擁有一定的資歷與經驗,工作也得心應手,但上面有比自己更資深的前輩壓著,身邊有隨時想超越你的同輩,下面又有一群「年輕就是本錢」、嫻熟科技的新世代員工虎視眈眈。
因此,大前研一建議,不管你是基層員工、還是擔任主管職,都要保持學習的習慣,隨時為自己的競爭力加值。因為,在全球化的時代,你不是跟中國人、美國人、日本人競爭,而是跟來自全球的頂尖人才競爭。他強調,學習跟智力高低無關,主要是取決於態度,以及培養獨立思考的能力。
該從哪方面打造個人競爭力?外語能力與使用網路的能力,在現今最為重要。
2.永遠做得比老闆要求的更多一點
只曉得「做好份內工作」的員工,等著被淘汰!因為,在這個競爭激烈的時代,有許多比你更積極的人,懂得永遠要比老闆要求的做更多。
威爾許強調,你必須超越上司對你的期待,讓他對你產生驚喜。別只等著上司傳授經驗、帶領你成長,事實上,你可以靠著自己的努力,提出能夠推動公司往前進的漂亮點子。
3.當個「用人達人型」主管
當你是員工時,你必須力求個人表現,以符合上司的要求;然而,當你成為上司,你的價值就不再來自個人成績,而是來自整個團隊每一個成員的表現。你必須了解部門中每個員工的特質,引導他們的潛能,幫助他們避免犯同樣的錯。
因此,你要讓自己成為知人善任的「用人達人」,帶動整個部門的整體成績,進而成為企業的重要競爭力。
4.隨時拓展人脈並懂得維繫
別以為只有負責某些職務的人需要人脈,事實上,不管你處於什麼位置,人脈關係永遠會帶給你更多意想不到的益處。
拓展人脈,處處是機會。除了特定活動的場合之外,從飛機上的鄰座到網際網路,再加上善用「朋友的朋友」,都是好管道。
人脈建立不難,重點在維繫。大前研一建議,最少一年一次,跟連絡簿、好友名單上的每一個人聊一下近況,保持住彼此的關係,讓對方一聽到你的名字就記起你。
5.勇敢邁向「繞道型」人生
一般人的人生,大抵不脫「求學→畢業→就職→結婚→升官→退休」的固定模式,踏著傳統上最多人走過的足跡。然而,大前研一卻認為,如果你還年輕,不妨跳脫這樣的模式,勇敢走一段「繞道」的人生。
大前研一以德國為例,許多大學生,會先休學一、兩年,趁著年輕到世界各地旅行,然後再回學校完成學業。或是畢業之後不馬上就業,而是先去旅行幾年。在每一趟旅程中,結交來自全球各國的朋友,開拓自己的視野與國際觀。
這樣,當你踏入職場,也許起步會比別人晚一點,但開闊的心胸與觀照全球的視野,會讓你比別人更加速進步,也擁有更多機會。繞道的人生,途中的各種經歷與美好風景,都會成為你衝刺的豐沛能量。
349期舵手的願景
在這瞬息萬變的時代,沒有任何一家企業敢保證可以永續經營。把視野往外看,也會發現外面的機會愈來愈多。
因此,除了在目前的位置打拚,也要時時問自己:「如果明天就離開現在的工作,我還能做什麼?手邊的存款,可以讓我活多久?」若你每次都能得到安心的答案,那麼,你就擁有著自信無虞的人生。
2006-06 天下雜誌 349期
你是正在力爭上游的基層員工、公司的當紅炸子雞、中高階主管、還是即將退休的資深元老?
無論你身在哪一個階段,當職場趨勢已從企業端的「終身雇用」轉變為個人端的「終身就業」,你必須時時增加自己的競爭力,即使二十年後,依然是企業搶著要的人才。
綜合日本趨勢專家大前研一、奇異前任執行長威爾許的觀點,以及《日經商業週刊》的報導,以下五件事,是你為自己加分的關鍵思考:
1.不管坐什麼位置,都要保持學習的習慣
出社會工作十年到十五年左右,會有一種「上下卡住」的閉塞感與無力感。因為,這個階段的上班族雖然擁有一定的資歷與經驗,工作也得心應手,但上面有比自己更資深的前輩壓著,身邊有隨時想超越你的同輩,下面又有一群「年輕就是本錢」、嫻熟科技的新世代員工虎視眈眈。
因此,大前研一建議,不管你是基層員工、還是擔任主管職,都要保持學習的習慣,隨時為自己的競爭力加值。因為,在全球化的時代,你不是跟中國人、美國人、日本人競爭,而是跟來自全球的頂尖人才競爭。他強調,學習跟智力高低無關,主要是取決於態度,以及培養獨立思考的能力。
該從哪方面打造個人競爭力?外語能力與使用網路的能力,在現今最為重要。
2.永遠做得比老闆要求的更多一點
只曉得「做好份內工作」的員工,等著被淘汰!因為,在這個競爭激烈的時代,有許多比你更積極的人,懂得永遠要比老闆要求的做更多。
威爾許強調,你必須超越上司對你的期待,讓他對你產生驚喜。別只等著上司傳授經驗、帶領你成長,事實上,你可以靠著自己的努力,提出能夠推動公司往前進的漂亮點子。
3.當個「用人達人型」主管
當你是員工時,你必須力求個人表現,以符合上司的要求;然而,當你成為上司,你的價值就不再來自個人成績,而是來自整個團隊每一個成員的表現。你必須了解部門中每個員工的特質,引導他們的潛能,幫助他們避免犯同樣的錯。
因此,你要讓自己成為知人善任的「用人達人」,帶動整個部門的整體成績,進而成為企業的重要競爭力。
4.隨時拓展人脈並懂得維繫
別以為只有負責某些職務的人需要人脈,事實上,不管你處於什麼位置,人脈關係永遠會帶給你更多意想不到的益處。
拓展人脈,處處是機會。除了特定活動的場合之外,從飛機上的鄰座到網際網路,再加上善用「朋友的朋友」,都是好管道。
人脈建立不難,重點在維繫。大前研一建議,最少一年一次,跟連絡簿、好友名單上的每一個人聊一下近況,保持住彼此的關係,讓對方一聽到你的名字就記起你。
5.勇敢邁向「繞道型」人生
一般人的人生,大抵不脫「求學→畢業→就職→結婚→升官→退休」的固定模式,踏著傳統上最多人走過的足跡。然而,大前研一卻認為,如果你還年輕,不妨跳脫這樣的模式,勇敢走一段「繞道」的人生。
大前研一以德國為例,許多大學生,會先休學一、兩年,趁著年輕到世界各地旅行,然後再回學校完成學業。或是畢業之後不馬上就業,而是先去旅行幾年。在每一趟旅程中,結交來自全球各國的朋友,開拓自己的視野與國際觀。
這樣,當你踏入職場,也許起步會比別人晚一點,但開闊的心胸與觀照全球的視野,會讓你比別人更加速進步,也擁有更多機會。繞道的人生,途中的各種經歷與美好風景,都會成為你衝刺的豐沛能量。
349期舵手的願景
在這瞬息萬變的時代,沒有任何一家企業敢保證可以永續經營。把視野往外看,也會發現外面的機會愈來愈多。
因此,除了在目前的位置打拚,也要時時問自己:「如果明天就離開現在的工作,我還能做什麼?手邊的存款,可以讓我活多久?」若你每次都能得到安心的答案,那麼,你就擁有著自信無虞的人生。
2011年7月8日 星期五
如何修改自己的英文文章
以下文章部分摘錄自研究生2.0 再加上自己的心得編修而成
要如何培養自我修改英文文章的能力呢?首先,你要先分清楚英文文章的錯誤。此書與寫作教學研究是將錯誤分成 global error 與 local error。global error 指的是較大範圍的錯誤,通常都比較嚴重甚至會影響到讀者的理解。local error 指的是在句子層面的錯誤,這種錯誤較不嚴重也比較不會影響對全篇的理解。以下我列出 global error 和 local error,讓大家更清楚。
Global errors:
1 Verb tenses
2 Verb forms
3 Modals
4 Conditional sentences
5 Passive voice
6 Relative, adverbial, and noun clauses
7 Sentence structure
8 Word order
9 Connecting words
Local errors:
1 Subject-verb agreement
2 Articles
3 SIngular and plural of nouns
4 Word choice
5 Word forms
6 Prepositions
要怎麼開始修改呢?一般來說,修改文章有兩種方式:一種是從頭讀到尾,邊讀邊改;另一種方式是焦點法,一次只看一種錯誤。對於剛開始練習修改的人,我個人覺得焦點法的效果會比較好,因為一次只改一種錯誤,比較容易看出有沒有錯誤。另外,像 verb tense 是需要全篇閱讀才能修改的,利用焦點法也比較容易改正這種錯誤。雖然使用焦點法可能花的時間較長,但我覺得效果很好。等以後熟悉如何改文章,就能邊讀邊改了。
在寫文章時,常出現的動詞時態錯誤有兩大類:用錯時態與不恰當轉換時態。
用錯時態
英文時態是挺不好學的,需要經過一段時間多聽多讀並試著自己使用,才有可能逐漸學會。對寫 paper 來說,其實沒有想像得困難。根據我自己的經驗與理解,對於大部分的內容來說,都是用過去式 (simple past) 和現在完成式 (present perfect)。一般說來,文獻探討開頭的前幾句,通常是用現在完成式,像是 Previous studies have demonstrated that …。到了分析各篇文章或特別說明某篇文章的發現時,多半用過去式。像是 Warschauer (1996) found that …。
但是到了 discussion 和 implication 時,常常是從單一研究的結果加上先前研究,希望能 generalize 到更大的範圍,這時候常常用的是簡單現在式 (simple present)。
只要掌握這些原則,就可以將用錯時態這個錯誤降到最低。
不恰當轉換時態
不恰當轉換時態的錯誤最常出現在長句中,前面與後面的時態不一致。拿書上的例子來說:
Although this is my first year in college, I have already found that there were some differences between high school and college. One of the things I learned in college is that a person has to be independent.
有沒有發現哪裡錯誤?前面都是用現在式和現在完成式,所以到了後面也要一致,採用現在式和現在完成式。改完之後是:
I have already found that there are some differences between high school and college
One of the things I have learned in college is that a person has to be independent.
依據我個人經驗,不恰當轉換時態這種錯其實比用錯時態還難找出來。我的辦法是在前一篇 如何修改自己的英文文章?之一 說明了,一次只看文章的一個重點,所以從頭到尾先把文章的時態改一次,這樣會比較容易注意到是否轉換時態。
這次要說的是 global error 的第二項:verb forms,也就是跟動詞形態相關的。在進一步說到錯誤之前,要先了解各種動詞形態的英文。
an infinitive:不定詞 (e.g., to walk, to study)
a base form:原形動詞 (e.g., walk, study)
a gerund or a present participant:V+ing (walking)
a past participle:過去分詞 (walked, spoken)
a simple past form:簡單過去式 (walked, spoke)
a verb phrase:動詞短語,也就是主要動詞加上助動詞 (e.g., has been speaking, has spoken)
裡面說到一點,如果你常看美劇或是生活在英語國家,請別太相信自己的耳朵來學習英文動詞形態,因為常常會聽不清楚 (眉批:而且很多美國人也搞不清楚)。這部分我自己覺得是得靠讀文法書作練習來加強,之後靠閱讀學習。
下面是書中的七個pretest,有點挑戰性。如果我沒有輸入錯的話,下方每個句子都有一個文法錯誤,請將它挑出來並改正。
Mario chosed to live in the dormitory rather than in an apartment.
The hikers had walk approximately 10 miles when they decided to set up camp.
Sometimes I totally confuse about English grammar.
The company did clearly deserved to obtain a large research grant to continue their innovative research.
An effective speaker tries look directly at his or her audience.
A grant writer hopes to presenting a convincing argument that clearly shows the value of a piece of research.
After finish work, Margarita likes to work out in the gym for at least an hour.
至於上面的錯誤地方在哪,我就不公佈了,詳情書上都有,或是接著看下面動詞形態七大問題點再回來作這些,會更知道如何糾錯。在往下看之前,我強烈建議你自己先作作上面的pretest以診斷你的英文問題。
1. 主要動詞的形態錯誤 (規則變化和不規則變化)。
2. 過去分詞的形態錯誤 (忘了加ed或不規則變化)。
3. 主要動詞或形容詞的形態錯誤,應該 be + 過去分詞 (常發生在與情緒相關的動詞,如:satisfy, confuse)。
4. 助動詞和主要動詞同時用在一句裡,而不是採用助動詞加原型動詞的形式。
5. 在動詞後面跟隨的動詞形態錯誤 (要確認是加 Ving 或是 to V)。
6. 不定詞使用錯誤 (要確認一下是不定詞還是 to + Ving)。
7. 不正確的使用原形動詞,而非使用現在分詞或不定詞。
在這部分其實細節挺多,很難在部落格上分享,而且本系列文章不在於講解文法,而是提供一個修改的 checklist 或一些策略。對於要改文章的人,我有些建議:
1. 熟讀動詞的搭配,有些動詞後面加不定詞,如 agree, appear, attempt 等,有些動詞後面加現在分詞,如:admit, avoid, quit 等,有些動詞是可接不定詞或現在分詞,如:begin, continue, hate等,有些動詞後面要加原形,像 let, have 等。
2. 善用電子字典。如果動詞的搭配不熟悉,那就要善用電子字典,像是靈格斯詞霸、搭配詞詞典都是很好的資源。
3. 特別注意句中的動詞,只要句中動詞後面加上原形、不定詞、現在分詞,請務必確認這是正確格式。
要如何培養自我修改英文文章的能力呢?首先,你要先分清楚英文文章的錯誤。此書與寫作教學研究是將錯誤分成 global error 與 local error。global error 指的是較大範圍的錯誤,通常都比較嚴重甚至會影響到讀者的理解。local error 指的是在句子層面的錯誤,這種錯誤較不嚴重也比較不會影響對全篇的理解。以下我列出 global error 和 local error,讓大家更清楚。
Global errors:
1 Verb tenses
2 Verb forms
3 Modals
4 Conditional sentences
5 Passive voice
6 Relative, adverbial, and noun clauses
7 Sentence structure
8 Word order
9 Connecting words
Local errors:
1 Subject-verb agreement
2 Articles
3 SIngular and plural of nouns
4 Word choice
5 Word forms
6 Prepositions
要怎麼開始修改呢?一般來說,修改文章有兩種方式:一種是從頭讀到尾,邊讀邊改;另一種方式是焦點法,一次只看一種錯誤。對於剛開始練習修改的人,我個人覺得焦點法的效果會比較好,因為一次只改一種錯誤,比較容易看出有沒有錯誤。另外,像 verb tense 是需要全篇閱讀才能修改的,利用焦點法也比較容易改正這種錯誤。雖然使用焦點法可能花的時間較長,但我覺得效果很好。等以後熟悉如何改文章,就能邊讀邊改了。
在寫文章時,常出現的動詞時態錯誤有兩大類:用錯時態與不恰當轉換時態。
用錯時態
英文時態是挺不好學的,需要經過一段時間多聽多讀並試著自己使用,才有可能逐漸學會。對寫 paper 來說,其實沒有想像得困難。根據我自己的經驗與理解,對於大部分的內容來說,都是用過去式 (simple past) 和現在完成式 (present perfect)。一般說來,文獻探討開頭的前幾句,通常是用現在完成式,像是 Previous studies have demonstrated that …。到了分析各篇文章或特別說明某篇文章的發現時,多半用過去式。像是 Warschauer (1996) found that …。
但是到了 discussion 和 implication 時,常常是從單一研究的結果加上先前研究,希望能 generalize 到更大的範圍,這時候常常用的是簡單現在式 (simple present)。
只要掌握這些原則,就可以將用錯時態這個錯誤降到最低。
不恰當轉換時態
不恰當轉換時態的錯誤最常出現在長句中,前面與後面的時態不一致。拿書上的例子來說:
Although this is my first year in college, I have already found that there were some differences between high school and college. One of the things I learned in college is that a person has to be independent.
有沒有發現哪裡錯誤?前面都是用現在式和現在完成式,所以到了後面也要一致,採用現在式和現在完成式。改完之後是:
I have already found that there are some differences between high school and college
One of the things I have learned in college is that a person has to be independent.
依據我個人經驗,不恰當轉換時態這種錯其實比用錯時態還難找出來。我的辦法是在前一篇 如何修改自己的英文文章?之一 說明了,一次只看文章的一個重點,所以從頭到尾先把文章的時態改一次,這樣會比較容易注意到是否轉換時態。
這次要說的是 global error 的第二項:verb forms,也就是跟動詞形態相關的。在進一步說到錯誤之前,要先了解各種動詞形態的英文。
an infinitive:不定詞 (e.g., to walk, to study)
a base form:原形動詞 (e.g., walk, study)
a gerund or a present participant:V+ing (walking)
a past participle:過去分詞 (walked, spoken)
a simple past form:簡單過去式 (walked, spoke)
a verb phrase:動詞短語,也就是主要動詞加上助動詞 (e.g., has been speaking, has spoken)
裡面說到一點,如果你常看美劇或是生活在英語國家,請別太相信自己的耳朵來學習英文動詞形態,因為常常會聽不清楚 (眉批:而且很多美國人也搞不清楚)。這部分我自己覺得是得靠讀文法書作練習來加強,之後靠閱讀學習。
下面是書中的七個pretest,有點挑戰性。如果我沒有輸入錯的話,下方每個句子都有一個文法錯誤,請將它挑出來並改正。
Mario chosed to live in the dormitory rather than in an apartment.
The hikers had walk approximately 10 miles when they decided to set up camp.
Sometimes I totally confuse about English grammar.
The company did clearly deserved to obtain a large research grant to continue their innovative research.
An effective speaker tries look directly at his or her audience.
A grant writer hopes to presenting a convincing argument that clearly shows the value of a piece of research.
After finish work, Margarita likes to work out in the gym for at least an hour.
至於上面的錯誤地方在哪,我就不公佈了,詳情書上都有,或是接著看下面動詞形態七大問題點再回來作這些,會更知道如何糾錯。在往下看之前,我強烈建議你自己先作作上面的pretest以診斷你的英文問題。
1. 主要動詞的形態錯誤 (規則變化和不規則變化)。
2. 過去分詞的形態錯誤 (忘了加ed或不規則變化)。
3. 主要動詞或形容詞的形態錯誤,應該 be + 過去分詞 (常發生在與情緒相關的動詞,如:satisfy, confuse)。
4. 助動詞和主要動詞同時用在一句裡,而不是採用助動詞加原型動詞的形式。
5. 在動詞後面跟隨的動詞形態錯誤 (要確認是加 Ving 或是 to V)。
6. 不定詞使用錯誤 (要確認一下是不定詞還是 to + Ving)。
7. 不正確的使用原形動詞,而非使用現在分詞或不定詞。
在這部分其實細節挺多,很難在部落格上分享,而且本系列文章不在於講解文法,而是提供一個修改的 checklist 或一些策略。對於要改文章的人,我有些建議:
1. 熟讀動詞的搭配,有些動詞後面加不定詞,如 agree, appear, attempt 等,有些動詞後面加現在分詞,如:admit, avoid, quit 等,有些動詞是可接不定詞或現在分詞,如:begin, continue, hate等,有些動詞後面要加原形,像 let, have 等。
2. 善用電子字典。如果動詞的搭配不熟悉,那就要善用電子字典,像是靈格斯詞霸、搭配詞詞典都是很好的資源。
3. 特別注意句中的動詞,只要句中動詞後面加上原形、不定詞、現在分詞,請務必確認這是正確格式。
2011年7月7日 星期四
醫學統計
intraclass correlation coefficient(組內相關係數)
Explain the use of intraclass correlation in relation to inter-rater reliability.
組內相關係數用來測量施測者間信度。ICC可被表示為組間的變異和總變異的比值。Intraclass correlation (ICC) is used to measure inter-rater reliability. ICC may be conceptualized as the ratio of between-groups variance to total variance.
資料建立:ICC的目的是用來評論施測者間的影響和受測族群間的關係。Data setup:The purpose of ICC is to assess the inter-rater (column) effect in relation to the grouping (row) effect, using two-way ANOVA.
解釋:當受測目標間沒有差異時,ICC的值會接近1,顯示總變異全來自受測者自身的不同。Interpretation: ICC will approach 1.0 when there is no variance within targets, indicating total variation in measurements on the Likert scale is due solely to the target (ex., subject, neighborhood) variable.
模型:ICC會隨著以下幾種原因而改變:評估者是包括所有施測者或是由可能的施測者中隨機抽來的;受測者是否包括所有受測者或是只是由隨機抽樣選出;信度的驗證是建立在個別的評估者上或是所有評者的平均。Models: ICC varies depending on whether the judges are all judges of interest or are conceived as a random sample of possible judges, and whether all targets are rated or only a random sample, and whether reliability is to be measured based on individual ratings or mean ratings of all judges. Shrout and Fleiss (1979).
ICC公式由來:令A代表個案真實的改變,令B代表由施測者間信度不良所造成的誤差,則ICC=A/(A+B)。B是個案間差異(一群施測者施測同樣個案時所給的不同分數)的均方(一組數的平方的平均值),由ANOVA計算。個案間變異的均方則為(A的k倍+B)Derivation of the ICC formula, following Ebel (1951: 409-411): Let A be the true variance in subjects' ratings due to the normal expectation that different subjects will have true different scores on the rating variable. Let B be the error variance in subjects' ratings attributable to inter-rater unreliability. The intent of ICC is to form the ratio, ICC = A/(A + B). B is simply the mean-square estimate of within-subjects variance (variance in the ratings for a given subject by a group of raters), computed in ANOVA. The mean-square estimate of between-subjects variance equals k times A (the true component) plus B (the inter-rater error component), since each mean contains a true component and an error component.
將B用MSwithin、A用(MSbetween –B)/k表示(因為MSbetween = kA + B),則公式就可化為:
ICC = rI = (MSbetween - MSwithin)/( MSbetween + [k - 1] MSwithin)。
此時MSbtween 反應了不同的個案的真實分數也就不同。MSwithin則表示由施測者間的誤差造成的分數不同。k代表了(施測者人數/受測者人數)。
Given B = MSwithin, and given MSbetween = kA + B, substituting these equalities into the intended equation (ICC = A/[A+B]), the equation for ICC reduces to the formula for the most-used version of intraclass correlation (Haggard, 1958: 60) : ICC = rI = (MSbetween - MSwithin)/( MSbetween + [k - 1] MSwithin) where MSbetween is the mean-square estimate of between-subjects variance, reflecting the normal expectation that different subjects will have true different scores on the rating variable
MSwithin is the mean-square estimate of within-subjects variance, or error attributed to inter-rater unreliability in rating the same person or target (row). k is the number of raters/ratings per target (person, neighborhood, etc.) = number of columns.
當MSbetween 等於MSwithin時,ICC等於0,顯示此時分組方式不會對ICC造成影響。
ICC is 0 when within-groups variance equals between-groups variance, indicative of the grouping variable having no effect.
Explain the use of intraclass correlation in relation to inter-rater reliability.
組內相關係數用來測量施測者間信度。ICC可被表示為組間的變異和總變異的比值。Intraclass correlation (ICC) is used to measure inter-rater reliability. ICC may be conceptualized as the ratio of between-groups variance to total variance.
資料建立:ICC的目的是用來評論施測者間的影響和受測族群間的關係。Data setup:The purpose of ICC is to assess the inter-rater (column) effect in relation to the grouping (row) effect, using two-way ANOVA.
解釋:當受測目標間沒有差異時,ICC的值會接近1,顯示總變異全來自受測者自身的不同。Interpretation: ICC will approach 1.0 when there is no variance within targets, indicating total variation in measurements on the Likert scale is due solely to the target (ex., subject, neighborhood) variable.
模型:ICC會隨著以下幾種原因而改變:評估者是包括所有施測者或是由可能的施測者中隨機抽來的;受測者是否包括所有受測者或是只是由隨機抽樣選出;信度的驗證是建立在個別的評估者上或是所有評者的平均。Models: ICC varies depending on whether the judges are all judges of interest or are conceived as a random sample of possible judges, and whether all targets are rated or only a random sample, and whether reliability is to be measured based on individual ratings or mean ratings of all judges. Shrout and Fleiss (1979).
ICC公式由來:令A代表個案真實的改變,令B代表由施測者間信度不良所造成的誤差,則ICC=A/(A+B)。B是個案間差異(一群施測者施測同樣個案時所給的不同分數)的均方(一組數的平方的平均值),由ANOVA計算。個案間變異的均方則為(A的k倍+B)Derivation of the ICC formula, following Ebel (1951: 409-411): Let A be the true variance in subjects' ratings due to the normal expectation that different subjects will have true different scores on the rating variable. Let B be the error variance in subjects' ratings attributable to inter-rater unreliability. The intent of ICC is to form the ratio, ICC = A/(A + B). B is simply the mean-square estimate of within-subjects variance (variance in the ratings for a given subject by a group of raters), computed in ANOVA. The mean-square estimate of between-subjects variance equals k times A (the true component) plus B (the inter-rater error component), since each mean contains a true component and an error component.
將B用MSwithin、A用(MSbetween –B)/k表示(因為MSbetween = kA + B),則公式就可化為:
ICC = rI = (MSbetween - MSwithin)/( MSbetween + [k - 1] MSwithin)。
此時MSbtween 反應了不同的個案的真實分數也就不同。MSwithin則表示由施測者間的誤差造成的分數不同。k代表了(施測者人數/受測者人數)。
Given B = MSwithin, and given MSbetween = kA + B, substituting these equalities into the intended equation (ICC = A/[A+B]), the equation for ICC reduces to the formula for the most-used version of intraclass correlation (Haggard, 1958: 60) : ICC = rI = (MSbetween - MSwithin)/( MSbetween + [k - 1] MSwithin) where MSbetween is the mean-square estimate of between-subjects variance, reflecting the normal expectation that different subjects will have true different scores on the rating variable
MSwithin is the mean-square estimate of within-subjects variance, or error attributed to inter-rater unreliability in rating the same person or target (row). k is the number of raters/ratings per target (person, neighborhood, etc.) = number of columns.
當MSbetween 等於MSwithin時,ICC等於0,顯示此時分組方式不會對ICC造成影響。
ICC is 0 when within-groups variance equals between-groups variance, indicative of the grouping variable having no effect.
2011年7月4日 星期一
蛋白質定量-二維電泳
2D Quant kit (GE healthcare)
1. 0, 5. 10.15.20.25.30 uL BSA (2 mg/mL)for standard curve or1 uL 2D-sample
2. add 500 uL precipitant,vortex, put on the ice,for 2~3 min
3. add 500 uL co-precipitant, vortex, centrifugal 10,000 xg, for 5 min
4. Remove the supernatant, centrifugal 10,000 xg, for 1 min, as clean as possible.
5. add 400 uL dd Water+ 100 uL copper solution,
6. add 1mL color reagent mixture (A : B = 100 : 1) for 20 min
7. On sample, OD480, dd water as blank
1. 0, 5. 10.15.20.25.30 uL BSA (2 mg/mL)for standard curve or1 uL 2D-sample
2. add 500 uL precipitant,vortex, put on the ice,for 2~3 min
3. add 500 uL co-precipitant, vortex, centrifugal 10,000 xg, for 5 min
4. Remove the supernatant, centrifugal 10,000 xg, for 1 min, as clean as possible.
5. add 400 uL dd Water+ 100 uL copper solution,
6. add 1mL color reagent mixture (A : B = 100 : 1) for 20 min
7. On sample, OD480, dd water as blank
蛋白質沉澱
TCA/ ACETONE Protein precipitation
Solution A: 10 % TCA, 0.07 %2-mecraptoethanol (2-MET) / in 100 % acetone
Solution B: 0.07 % 2-MET, 1 mM PMSF/ in 80 % acetone
Store at -20~80 oC
1. Sample weight: Solution A = 1:10, store at -20 oC for 45 min (no more than 1 hr)
2. Centrifugal,12,000 rpm, 15 min, 4 oC, remove the supernatant
3. Solution B wash x 3~5 times
4. 100 % Acetone wash x 1, As clean as possible
5. Air dry for 5 min
6. 100 uL rehydration buffer
Solution A: 10 % TCA, 0.07 %2-mecraptoethanol (2-MET) / in 100 % acetone
Solution B: 0.07 % 2-MET, 1 mM PMSF/ in 80 % acetone
Store at -20~80 oC
Rehydration buffer
8M urea. 2 % CHAPS, 0.5% IPG buffer, 0.002% Bromophenol blue
When need to use, add 20 mM (0.0031 g/mL DTT)
Solution A: 10 % TCA, 0.07 %2-mecraptoethanol (2-MET) / in 100 % acetone
Solution B: 0.07 % 2-MET, 1 mM PMSF/ in 80 % acetone
Store at -20~80 oC
1. Sample weight: Solution A = 1:10, store at -20 oC for 45 min (no more than 1 hr)
2. Centrifugal,12,000 rpm, 15 min, 4 oC, remove the supernatant
3. Solution B wash x 3~5 times
4. 100 % Acetone wash x 1, As clean as possible
5. Air dry for 5 min
6. 100 uL rehydration buffer
Solution A: 10 % TCA, 0.07 %2-mecraptoethanol (2-MET) / in 100 % acetone
Solution B: 0.07 % 2-MET, 1 mM PMSF/ in 80 % acetone
Store at -20~80 oC
Rehydration buffer
8M urea. 2 % CHAPS, 0.5% IPG buffer, 0.002% Bromophenol blue
When need to use, add 20 mM (0.0031 g/mL DTT)
2011年6月29日 星期三
抽取 RNA 與RNA 電泳
1. The following gel electrophoresis conditions are recommended:
- use 1X TAE buffer instead of 1X TBE
- agarose 1.1 %~1.2 %
- add ethidium bromide (EtBr) to the gel and electrophoresis buffer to avoid the additional (potentially RNAse-prone) step of gel staining
- always use fresh gel and buffer as well as clean electrophoresis equipment for RNA analysis. Wear gloves to protect RNA samples from degradation by nucleases and avoid a hand contact with EtBr.
- use running voltage up to 10 V/cm (10V per each cm of space between the electrodes in electrophoretic chamber). Do not use high voltage to avoid RNA degradation during electrophoresis.
2. Heat an aliquot of the RNA solution at 70°C for 1 min and place it on ice before loading on a gel.
3. Load a known amount of DNA or RNA ladder alongside your RNA sample as a standard for determining the RNA concentration. RNA concentration can be roughly estimated assuming that the efficiency of EtBr incorporation in rRNA is the same as for DNA (the ribosomal RNA may be considered a double-stranded molecule due to its extensive secondary structure).
4. The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA bands and extending to the area of shorter fragments. RNA showing this extent of degradation is still good for further procedures. However, if the downward smearing is so pronounced that the rRNA bands do not have a discernible lower edge, this RNA should be discarded.
The following characteristics indicate successful RNA preparation:
- For mammalian total RNA, two intensive bands at approximately 4.5 and 1.9 kb should be observed against a light smear. These bands represent 28S and 18S rRNA. The ratio of intensities of these bands should be about 1.5-2.5:1. Intact mammalian poly (A)+ RNA appears as a smear sized from 0.1 to 4-7 (or more) kb with faint 28S and 18S rRNA bands.
- In the case of RNA from non-mammalian sources (plants, insects, yeast, amphibians), the normal mRNA smear on the non-denaturing agarose gel may not exceed 2-3 kb. Moreover, the overwhelming majority of invertebrates have 28s rRNA with a so-called "hidden break" (Ishikawa, 1977). In some organisms the interaction between the parts of 28s rRNA is rather weak, so the total RNA preparation exhibits a single 18s-like rRNA band even on a non-denaturing gel. In other species the 28s rRNA is more robust, so it is still visible as a second band.
Note: If your experimental RNA is shorter than expected and/or degraded according to electrophoresis data, prepare fresh RNA after checking the quality of RNA purification reagents. If problems persist, you may need to find another source of tissue/cells. In some cases, partially degraded RNA is only available (e.g. tumor samples or hard treated tissues). This RNA can be used for cDNA preparation, however the cDNA sample will contain reduced number of full-length molecules.
- Commonly, genomic DNA contamination does not exceed the amount seen on the agarose/EtBr gel as a weak band of high molecular weight. Such contamination does not affect cDNA synthesis. DNase treatment to degrade genomic DNA is not recommended. In some cases, excess of genomic DNA can be removed by LiCl precipitation or by phenol:chloroform extraction.
http://www.ambion.com/techlib/append/supp/rna_gel.html
http://www.protocol-online.org/prot/Molecular_Biology/RNA/RNA_Electrophoresis/index.html
10X DNA/RNA loading dye
Glycerol 50%
Bromphenol Blue 0.4%
Xylene Cyanol 0.4%
- use 1X TAE buffer instead of 1X TBE
- agarose 1.1 %~1.2 %
- add ethidium bromide (EtBr) to the gel and electrophoresis buffer to avoid the additional (potentially RNAse-prone) step of gel staining
- always use fresh gel and buffer as well as clean electrophoresis equipment for RNA analysis. Wear gloves to protect RNA samples from degradation by nucleases and avoid a hand contact with EtBr.
- use running voltage up to 10 V/cm (10V per each cm of space between the electrodes in electrophoretic chamber). Do not use high voltage to avoid RNA degradation during electrophoresis.
2. Heat an aliquot of the RNA solution at 70°C for 1 min and place it on ice before loading on a gel.
3. Load a known amount of DNA or RNA ladder alongside your RNA sample as a standard for determining the RNA concentration. RNA concentration can be roughly estimated assuming that the efficiency of EtBr incorporation in rRNA is the same as for DNA (the ribosomal RNA may be considered a double-stranded molecule due to its extensive secondary structure).
4. The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA bands and extending to the area of shorter fragments. RNA showing this extent of degradation is still good for further procedures. However, if the downward smearing is so pronounced that the rRNA bands do not have a discernible lower edge, this RNA should be discarded.
The following characteristics indicate successful RNA preparation:
- For mammalian total RNA, two intensive bands at approximately 4.5 and 1.9 kb should be observed against a light smear. These bands represent 28S and 18S rRNA. The ratio of intensities of these bands should be about 1.5-2.5:1. Intact mammalian poly (A)+ RNA appears as a smear sized from 0.1 to 4-7 (or more) kb with faint 28S and 18S rRNA bands.
- In the case of RNA from non-mammalian sources (plants, insects, yeast, amphibians), the normal mRNA smear on the non-denaturing agarose gel may not exceed 2-3 kb. Moreover, the overwhelming majority of invertebrates have 28s rRNA with a so-called "hidden break" (Ishikawa, 1977). In some organisms the interaction between the parts of 28s rRNA is rather weak, so the total RNA preparation exhibits a single 18s-like rRNA band even on a non-denaturing gel. In other species the 28s rRNA is more robust, so it is still visible as a second band.
Note: If your experimental RNA is shorter than expected and/or degraded according to electrophoresis data, prepare fresh RNA after checking the quality of RNA purification reagents. If problems persist, you may need to find another source of tissue/cells. In some cases, partially degraded RNA is only available (e.g. tumor samples or hard treated tissues). This RNA can be used for cDNA preparation, however the cDNA sample will contain reduced number of full-length molecules.
- Commonly, genomic DNA contamination does not exceed the amount seen on the agarose/EtBr gel as a weak band of high molecular weight. Such contamination does not affect cDNA synthesis. DNase treatment to degrade genomic DNA is not recommended. In some cases, excess of genomic DNA can be removed by LiCl precipitation or by phenol:chloroform extraction.
http://www.ambion.com/techlib/append/supp/rna_gel.html
http://www.protocol-online.org/prot/Molecular_Biology/RNA/RNA_Electrophoresis/index.html
10X DNA/RNA loading dye
Glycerol 50%
Bromphenol Blue 0.4%
Xylene Cyanol 0.4%
2011年6月27日 星期一
[學術論文]論文時勢
以下部分內容出自研究生2.0
進行部分編修 純做自己領悟學習用 絕無侵犯版權之意圖
欲知詳細內容 請至http://newgenerationresearcher.blogspot.com/2011/06/blog-post.html
論文基本文法概念
對所有的研究者或學者們來說,在論文的不同區塊使用正確的時態是一件相當挑戰的任務
當提到「自己的論文文章本身」
任何時候提及自己的論文本身,包括提及論文中的章節或圖表時,皆應使用現在簡單式。
當提到「一般事實」
「事實」表示恆常不變之真理。因此當論文中提及恆為事實之事件或現象,應使用現在簡單式。
當提到「當前研究現象與成果」
當論文中提及該領域「當前研究現象與成果」,表示過去到現在該領域累積的研究成果,應使用現在完成式, 但不包含提及特定文獻,以下會加以說明。
當提到「我們的研究」
我們的研究工作(非指文章本身)是在過去完成的,所以應使用過去式。但切記要與研究方法 (methods) 與研究成果 (results) 區隔,研究成果可視為「事實」,因此需使用現在式。
當提到「特定作者及其文獻」
當提到其他作者的特定某篇文獻,這些作者們同樣是在過去完成他們的研究工作,因此,過去的研究工作使用過去式
當提到「未來研究方向」
除非我們已有具體的未來研究方向計畫,不然請勿使用未來式來描述未來研究的方向,或是可換個方式來表達,如 something has to be done。
時態練習題解說
以下提供時態練習題的解說,請先完成練習,在閱讀以下解說,能幫助您迅速瞭解撰寫論文時應選擇何種時態:
1. This aim of this paper (was/ is/ has been/ will be) to identify the factors . . .
本句常出現在摘要中,來概括「本論文」的目的,而非本研究,提到自己本身的文章 (this paper) 時使用現在式最佳,故本題應選 is。
2. Previous researchers (identify/ identified/ have identified) a number of factors.
本句為一般性的描述當前的研究現象與成果 (previous researchers,非指特定作者),應使用現在完成式, 故本題應選 have identified。
3. We (determine/ determined/ have determined) that the factors . . .
本句表達我們在研究的過程中判定/決定某事,這個動作是在過去完成的,應使用過去式,故本題應選 determined。
4. Pierce (claims/ claimed/ has claimed) that the most significant factors include . . .
本句明確的指向特定的作者 (Pierce),因此為過去之研究,應使用過去式,故本題應選 claimed。
5. It is commonly understood that such factors (will be/ are/ were/ have been) crucial to . . .
本句開頭 (commonly understood...)表明後面陳述的為眾人熟知的事實,應使用現在式,故本題應選 are。
6. Figure 7 (shows/ showed/ has shown) that that these factors (were/ are/ have been/ will be) important under these conditions.
本句提及論文中的圖表 (Figure 7),當提到我們論文本身或其中的章節及圖表,應使用現在簡單式,故應選 shows。
7. Despite these findings, further investigation (is/ was/ will be/ has been) necessary.
論文寫作中請避免使用未來式。我們的論文在未來五年內都有被人閱讀的機會,所以使用未來式將很難界定時間。因此,使用現在式較為安全,故本題應選 is。
8. In the following section, we (discussed/ will discuss/ discuss) these implications.
本句提及我們的文章中某一章節 (in the following section),應使用現在簡單式,故本題應選 discuss。
9. Recent advances (enabled/ have enabled/ enable/ will enable) further development of these resources.
本句首的Recent 為關鍵字,表示自過去某段時間一直到現在的成果,應使用現在完成,故本題應選 have enabled。
10. In a recent study, Lee et al. (have determined/ determined/ determines) that these factors . . .
雖然本句開頭與上一句相似,但後面明確指出某特定文獻 (Lee et al.),因此選用過去式,故本題應選 determined。
進行部分編修 純做自己領悟學習用 絕無侵犯版權之意圖
欲知詳細內容 請至http://newgenerationresearcher.blogspot.com/2011/06/blog-post.html
論文基本文法概念
對所有的研究者或學者們來說,在論文的不同區塊使用正確的時態是一件相當挑戰的任務
當提到「自己的論文文章本身」
任何時候提及自己的論文本身,包括提及論文中的章節或圖表時,皆應使用現在簡單式。
當提到「一般事實」
「事實」表示恆常不變之真理。因此當論文中提及恆為事實之事件或現象,應使用現在簡單式。
當提到「當前研究現象與成果」
當論文中提及該領域「當前研究現象與成果」,表示過去到現在該領域累積的研究成果,應使用現在完成式, 但不包含提及特定文獻,以下會加以說明。
當提到「我們的研究」
我們的研究工作(非指文章本身)是在過去完成的,所以應使用過去式。但切記要與研究方法 (methods) 與研究成果 (results) 區隔,研究成果可視為「事實」,因此需使用現在式。
當提到「特定作者及其文獻」
當提到其他作者的特定某篇文獻,這些作者們同樣是在過去完成他們的研究工作,因此,過去的研究工作使用過去式
當提到「未來研究方向」
除非我們已有具體的未來研究方向計畫,不然請勿使用未來式來描述未來研究的方向,或是可換個方式來表達,如 something has to be done。
時態練習題解說
以下提供時態練習題的解說,請先完成練習,在閱讀以下解說,能幫助您迅速瞭解撰寫論文時應選擇何種時態:
1. This aim of this paper (was/ is/ has been/ will be) to identify the factors . . .
本句常出現在摘要中,來概括「本論文」的目的,而非本研究,提到自己本身的文章 (this paper) 時使用現在式最佳,故本題應選 is。
2. Previous researchers (identify/ identified/ have identified) a number of factors.
本句為一般性的描述當前的研究現象與成果 (previous researchers,非指特定作者),應使用現在完成式, 故本題應選 have identified。
3. We (determine/ determined/ have determined) that the factors . . .
本句表達我們在研究的過程中判定/決定某事,這個動作是在過去完成的,應使用過去式,故本題應選 determined。
4. Pierce (claims/ claimed/ has claimed) that the most significant factors include . . .
本句明確的指向特定的作者 (Pierce),因此為過去之研究,應使用過去式,故本題應選 claimed。
5. It is commonly understood that such factors (will be/ are/ were/ have been) crucial to . . .
本句開頭 (commonly understood...)表明後面陳述的為眾人熟知的事實,應使用現在式,故本題應選 are。
6. Figure 7 (shows/ showed/ has shown) that that these factors (were/ are/ have been/ will be) important under these conditions.
本句提及論文中的圖表 (Figure 7),當提到我們論文本身或其中的章節及圖表,應使用現在簡單式,故應選 shows。
7. Despite these findings, further investigation (is/ was/ will be/ has been) necessary.
論文寫作中請避免使用未來式。我們的論文在未來五年內都有被人閱讀的機會,所以使用未來式將很難界定時間。因此,使用現在式較為安全,故本題應選 is。
8. In the following section, we (discussed/ will discuss/ discuss) these implications.
本句提及我們的文章中某一章節 (in the following section),應使用現在簡單式,故本題應選 discuss。
9. Recent advances (enabled/ have enabled/ enable/ will enable) further development of these resources.
本句首的Recent 為關鍵字,表示自過去某段時間一直到現在的成果,應使用現在完成,故本題應選 have enabled。
10. In a recent study, Lee et al. (have determined/ determined/ determines) that these factors . . .
雖然本句開頭與上一句相似,但後面明確指出某特定文獻 (Lee et al.),因此選用過去式,故本題應選 determined。
[實驗技術]西方點墨法
General Western blot
Makes 15 ml of 5x Loading Dye
3ml of 20% SDS
3.75 mL 1M Tris - pH 6.8
9 mg bromphenol blue
1.16 gm DTT (Alternatively add 2.4ml B-mercaptoethanol)
Add water to a final volume of 10.5ml
Mix the above reagents well before adding glycerol
4.5 mL Glycerol
Mix well again Store at 4 degrees
Running Buffer (1L)
Glycine 14.4 g
Tris 3.03 g
10% SDS 10 mL
Running condition
50V for 10~15 min
100V for 90 min
Transfer buffer (1 L)
Glycine 14.4 g
Tris 3.03 g
Methanol 100 mL
Transfer condition
200 mA/set for 120 min
blocking 5% skim milk / TBST
10X TBS Buffer (Tris buffer saline) (1 liter)
Tris Base [121.14] 500mM 60.5 g
Sodium Chloride (NaCl) 1.5 M 87.6 g
Add dH2O dilute to 1L pH= 7.5(以HCl滴定pH)
TBST (TBS + 0.05% Tween 20)
一抗 O/N, TBST 5 min x 6,
二抗 不超過2hr, TBST, x 6
Strip, ddH2O, microwave for 5 mins
Blocking 10% skim milk / TBST O/N 進行下一個階段的實驗
2011年6月26日 星期日
[實驗技術]細胞週期染色( propidium iodide stain)
流式細胞儀之應用
細胞週期染色(flow cytometry propidium iodide stain)
在細胞凋亡的早期,發現有染色體濃縮 (chromosome condensation)的情況,染色質會聚集成半月狀沿著核膜周圍分佈,以及出現細胞核萎縮 (pyknosis); 晚期時細胞膜皺褶、失去原有細胞骨架但胞器仍在,胞膜及胞漿保持完整,去氧核糖核酸 (DNA)會斷裂成特定大小片段 (180-200 base pair) ,以及有凋亡小體 (apoptosis bodies)的產生,最後被鄰近的細胞清除。propidium iodide是一種染劑,中文名為碘化丙啶。能夠插入DNA或RNA鹼基對中並在受到488 nm激發後放出在562-588 nm間波長的激發光,因此我們可以利用這種特性去針對那些正在凋亡或染色體受損斷裂的細胞做標定並偵測它的含量。
材料和方法
1. 待測細胞固定染色方法如下(酒精固定法及PI 染色法):
(1) 置備懸浮細胞液,調整至濃度約5x 10^6 cells/ml。若細胞株為attached cell line,先以 Trypsin 將細胞打下,調整細胞濃度(注意trypsin 處理不可過度,細胞會破裂)。
(2) 取 1 mL 細胞液,以冰冷的 PBS buffer 清洗細胞一次,離心後,去除上清液。
(3) 以剩餘的上清液將細胞打散(必須確定細胞完全打散)。
(4) 在震盪器上(轉速不可開太快)一邊震盪一邊逐一滴入3 ml 70% 冰冷的酒精(注意觀察細胞,不要讓細胞發生凝集現象)。
(5) 置於 4度 固定至少一小時。(正常處理妥善,可以放至少兩個禮拜)
(6) 染色前,將細胞從 4 度取出,以 300g 離心5 分鐘,去除上清液。
(7) 以剩餘的上清液將細胞打散,加入 5 ml PBS buffer,靜置 3 分鐘後離心,去除上清液。
(8) 重複步驟7,以5 ml PBS buffer 再清洗細胞一次。
(9) 加入 1 ml PI/Triton X-100(終濃度 PI= 20ug/ml, Triton-X 100=0.1%, RNase A= 0.2mg/ml),均勻打散細胞(這步驟相當重要,以免上機時細胞卡管),避光染色至少30 分鐘。
(10) 上機前打散細胞並以 35um 尼龍篩網過濾樣本。
(11) 陽性控制組(已經固定而且以PI 染色的健康細胞)
(12) 陰性控制組 (已經固定,沒有以PI染色,可以調整細胞大小聚落,並可確認是否染色成功)
儀器設定
1. 參數選擇
要選擇的參數有:FS Lin、SS Lin、FL Lin、FL Peak、Ratio(Ratio= FL Peak/ FL Lin)
2. 門檻值設定
門檻值可設在FS 或FL
3. 分析圖形
對動物細胞進行分析,先利用陽性控制組細胞調整設定及繪圖。第一張要看的圖就是FS Lin- SS Lin,先調整FS、SS 電壓及Gain 值,使細胞群落在FS Lin- SS Lin 圖形中央,圈選所要觀察的細胞群,於 FL Lin- Ratio、FL Lin- FL Peak 圖形調整FL 電壓及Gain 值設定,區分螢光來源是單顆細胞還是聚集黏在一起的細胞(與基因組及倍體數分析的方法相同)。在FL Lin- Ratio 或FL Lin- FL Peak 圈選單顆細胞,於FL Lin histogram 分析單顆細胞的螢光訊號。
參考文獻出自BACKMAN FLOW COULTER EPICS XL 使用手冊
FL1:525 nM (FITC, GFP) 綠
FL2:576 nM (PE,YFP) 黃
FL3:620 nM (ECD,PI) 橘
FL4:675 nM (PC5, 7-ADD) 紅
FL5: 755 nM (PC7, PE-CY7) 紫
PI(Propidium iodide,Sigma,MO,USA)stain之原理,為PI染劑可染雙股之核酸鏈,當其以嵌合的方式(intercalation)與雙股核酸鏈結合會產生紅色螢光(640nm),但因其也會與雙股的RNA鏈相結合,故當用於測量細胞DNA時必須將細胞內之RNA移除,可藉由加入RNase達成此目的。
細胞週期染色(flow cytometry propidium iodide stain)
在細胞凋亡的早期,發現有染色體濃縮 (chromosome condensation)的情況,染色質會聚集成半月狀沿著核膜周圍分佈,以及出現細胞核萎縮 (pyknosis); 晚期時細胞膜皺褶、失去原有細胞骨架但胞器仍在,胞膜及胞漿保持完整,去氧核糖核酸 (DNA)會斷裂成特定大小片段 (180-200 base pair) ,以及有凋亡小體 (apoptosis bodies)的產生,最後被鄰近的細胞清除。propidium iodide是一種染劑,中文名為碘化丙啶。能夠插入DNA或RNA鹼基對中並在受到488 nm激發後放出在562-588 nm間波長的激發光,因此我們可以利用這種特性去針對那些正在凋亡或染色體受損斷裂的細胞做標定並偵測它的含量。
材料和方法
1. 待測細胞固定染色方法如下(酒精固定法及PI 染色法):
(1) 置備懸浮細胞液,調整至濃度約5x 10^6 cells/ml。若細胞株為attached cell line,先以 Trypsin 將細胞打下,調整細胞濃度(注意trypsin 處理不可過度,細胞會破裂)。
(2) 取 1 mL 細胞液,以冰冷的 PBS buffer 清洗細胞一次,離心後,去除上清液。
(3) 以剩餘的上清液將細胞打散(必須確定細胞完全打散)。
(4) 在震盪器上(轉速不可開太快)一邊震盪一邊逐一滴入3 ml 70% 冰冷的酒精(注意觀察細胞,不要讓細胞發生凝集現象)。
(5) 置於 4度 固定至少一小時。(正常處理妥善,可以放至少兩個禮拜)
(6) 染色前,將細胞從 4 度取出,以 300g 離心5 分鐘,去除上清液。
(7) 以剩餘的上清液將細胞打散,加入 5 ml PBS buffer,靜置 3 分鐘後離心,去除上清液。
(8) 重複步驟7,以5 ml PBS buffer 再清洗細胞一次。
(9) 加入 1 ml PI/Triton X-100(終濃度 PI= 20ug/ml, Triton-X 100=0.1%, RNase A= 0.2mg/ml),均勻打散細胞(這步驟相當重要,以免上機時細胞卡管),避光染色至少30 分鐘。
(10) 上機前打散細胞並以 35um 尼龍篩網過濾樣本。
(11) 陽性控制組(已經固定而且以PI 染色的健康細胞)
(12) 陰性控制組 (已經固定,沒有以PI染色,可以調整細胞大小聚落,並可確認是否染色成功)
儀器設定
1. 參數選擇
要選擇的參數有:FS Lin、SS Lin、FL Lin、FL Peak、Ratio(Ratio= FL Peak/ FL Lin)
2. 門檻值設定
門檻值可設在FS 或FL
3. 分析圖形
對動物細胞進行分析,先利用陽性控制組細胞調整設定及繪圖。第一張要看的圖就是FS Lin- SS Lin,先調整FS、SS 電壓及Gain 值,使細胞群落在FS Lin- SS Lin 圖形中央,圈選所要觀察的細胞群,於 FL Lin- Ratio、FL Lin- FL Peak 圖形調整FL 電壓及Gain 值設定,區分螢光來源是單顆細胞還是聚集黏在一起的細胞(與基因組及倍體數分析的方法相同)。在FL Lin- Ratio 或FL Lin- FL Peak 圈選單顆細胞,於FL Lin histogram 分析單顆細胞的螢光訊號。
參考文獻出自BACKMAN FLOW COULTER EPICS XL 使用手冊
FL1:525 nM (FITC, GFP) 綠
FL2:576 nM (PE,YFP) 黃
FL3:620 nM (ECD,PI) 橘
FL4:675 nM (PC5, 7-ADD) 紅
FL5: 755 nM (PC7, PE-CY7) 紫
PI(Propidium iodide,Sigma,MO,USA)stain之原理,為PI染劑可染雙股之核酸鏈,當其以嵌合的方式(intercalation)與雙股核酸鏈結合會產生紅色螢光(640nm),但因其也會與雙股的RNA鏈相結合,故當用於測量細胞DNA時必須將細胞內之RNA移除,可藉由加入RNase達成此目的。
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[論文寫作]常用連接詞
一、And 表示並列關係 (and)in addition/and/similarly/likewise/as well as/besides/furthermore/also/moreover/too/not only ... but/even/besides this/...
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Aim,goal,purpose,end,target,object,objective 這些名词均有“目標、目的”之意。 寫作時,要注意區分語意: Aim : 表示你有計畫要完成特定目標(具體而明確的),通常是指短期目標 Goal:通常是指球賽或比賽的得...
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[資料來源:網路] Method指做某件工作的固定模式,而且強調以效率和準確性為目的,是一種固定而且無需變化的方法。 Way指單一技巧或整個操作過程,指具有某種思路或風格的方法。對於方法或是過程本身是好是壞,不做任何評價。 Approach是学習或研究問题的方法,指解...