avatar
v*e
2
最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
不幸。
dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
LATER里比较难剥,因为变硬了。
然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
最后用RNAEASY纯化,
大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢
avatar
m*n
3
这个称谓娱乐界基本都知道和接受的吧。 百度“谭咏麟为什么叫校长”。以下内容是
转帖,但应该基本属实(具体是哪一年哪场演唱会说出来的不清楚)。第二段属歌迷主
观感受,不评论。
----------
谭咏麟并不是哪个学校的校长,他之所以被叫作谭校长,是自从84年开始每逢开演唱
会即连开数十场,整个月每天晚上都是如此,于是谭咏麟在86年20场万众狂欢演唱会的
某一场上亲口对全场歌迷说:“整个红馆就象一所万人大学校,你们每天都要来上夜校
,而我每天晚上都要和你们在一起,用歌教大家一些做人的道理,我觉得自已就象这所
学校的校长啊!”,于是,第二天,校长的称号就流行风靡至今了!
另外就是“校长”是一个学校里最大的,谭咏麟是香港乐坛最大的,其他的歌手都像
是他的学生一样,和他不是一个档次上的。
------
小纠一下,以我的感觉,应该没有“第二天,校长的称号就流行...". 感觉以前大家还
是习惯叫阿伦之类的。最近几年年纪大了,其他称呼就觉得没有这个称呼更合适了。反
正就是好提携个后辈,后辈也给他个面子,就叫校长。
最近流行有关”校长“丑闻的说法,适逢谭校长在长沙(还是哪儿)开演唱会,有个无
良记者在自己的微博上说,谭咏麟演唱会的第一句就说”以后不要再叫我校长“。这话
是瞎说的,被谭咏麟看到了,他的回复是 “我懷疑這位傳媒朋友有沒有入場觀看演出
?雖然近日有禽獸校長出現但大部份從事教育界的校長都是盡心盡力的,不應抹殺他們
的功勞!。 ”
就这样。校长的称谓被某些变态妖魔化了。但此校长不是彼校长。
【 在 viamedia (Mens conscia recti) 的大作中提到: 】
avatar
v*e
4
有人知道吗?

【在 v****e 的大作中提到】
: 最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
: 不幸。
: dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
: LATER里比较难剥,因为变硬了。
: 然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
: 最后用RNAEASY纯化,
: 大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢

avatar
w*w
5
百度的很全面~~

【在 m********n 的大作中提到】
: 这个称谓娱乐界基本都知道和接受的吧。 百度“谭咏麟为什么叫校长”。以下内容是
: 转帖,但应该基本属实(具体是哪一年哪场演唱会说出来的不清楚)。第二段属歌迷主
: 观感受,不评论。
: ----------
: 谭咏麟并不是哪个学校的校长,他之所以被叫作谭校长,是自从84年开始每逢开演唱
: 会即连开数十场,整个月每天晚上都是如此,于是谭咏麟在86年20场万众狂欢演唱会的
: 某一场上亲口对全场歌迷说:“整个红馆就象一所万人大学校,你们每天都要来上夜校
: ,而我每天晚上都要和你们在一起,用歌教大家一些做人的道理,我觉得自已就象这所
: 学校的校长啊!”,于是,第二天,校长的称号就流行风靡至今了!
: 另外就是“校长”是一个学校里最大的,谭咏麟是香港乐坛最大的,其他的歌手都像

avatar
C*h
6
液氮加Trizol,RNA不会降解。
但是,你的RNA很可能是在剥的10分钟,降解了。
所以,你现在就是要尽量想办法,缩短“剥”的这一个过程。

【在 v****e 的大作中提到】
: 最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
: 不幸。
: dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
: LATER里比较难剥,因为变硬了。
: 然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
: 最后用RNAEASY纯化,
: 大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢

avatar
l*s
7
我觉得不会这么快,磷酸化带白还好说。RNA不是这么不稳定的
avatar
v*e
8
谢谢!我正在试不同的方法!

【在 v****e 的大作中提到】
: 最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
: 不幸。
: dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
: LATER里比较难剥,因为变硬了。
: 然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
: 最后用RNAEASY纯化,
: 大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢

avatar
q*8
9
你是提完以后立马降解的,还是刚提完还可以,-80放了几天以后才不行的?
avatar
g*a
10
RNA不至于这么快降解吧,觉得你操作没问题啊,我们有时候取组织种类比较多,还要
分别称重,十分钟一个老鼠还算快的,是不是后面抽提的问题或者trizol出问题了?X
avatar
I*a
11
是qiagen的RNaesy kit?
avatar
D*a
12
马后炮一句。。这种实验好像先做做试试流程再上真老鼠比较好吧。
avatar
D*a
13
楼主也可以说说什么组织大家看看有什么解剖窍门。
avatar
v*e
14
是dorsal-lateral prostate!做regular qPCR没问题!可是做quality check for
microarray 说RNA有降解!

【在 v****e 的大作中提到】
: 最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
: 不幸。
: dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
: LATER里比较难剥,因为变硬了。
: 然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
: 最后用RNAEASY纯化,
: 大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢

avatar
C*u
15
Using more trizol might help!

【在 v****e 的大作中提到】
: 最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
: 不幸。
: dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
: LATER里比较难剥,因为变硬了。
: 然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
: 最后用RNAEASY纯化,
: 大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢

avatar
M*e
16
我的办法可能能帮你解决问题.
我曾经试过多种办法提没有降解(用Bio-Rad or Agilent Bioanalyzer测integrity or
quality (RNA Integrity Number (RIN) by Agilent or RNA Quality Index (RQI)
by Bio-Rad),不是仅仅用Nanodrop测RNA integrity or quality)的total RNA,然后
做microarray.
1.液氮研磨:RNA quality好,可以做microarray,但是麻烦和太慢(原因你应该知道
)。
2. Glass tissue homogenizer: it didn't work for me to grind tissue in
Trizol in ice bucket with ice, although my tissue was thin and tiny.
Bioanalyzer showed RNA degradation.It's also low through-put.
3. Hand-held electronic homogenizer: it did not apply in my case, since my
tissue was thin and tiny.It got entrapped or lost easily.
4. Used RNALater to treat my tissue first before RNA extraction. As you know
, RNALater hardens tissue by dehydration and it makes tissue gooey. I found
that RNALater did not work for me. Bioanalyzer showed that my total RNA
still underwent degradation in my very thin and tiny tissue treated with
RNALater.
我使用RNALater处理过小而且薄的mouse tissue,我发现RNALater不能很好地防止RNA降
解。Bioanalyzer显示:我的total RNA有相当严重的降解,所以,我认为RNALater没有
像宣传的那么好。
我反对相信:只要用RNALater处理tissue,RNA就一定不怎么降解。
如果你先用RNALater处理tissue, 然后将处理过的tissue马上放在液氮中,那使用
RNALater就是多此一举。
5. Eventually, I came up an easy and relatively high through-put way to
reliably extract total RNA from mouse tissue.
a. harvest your tissue as quickly as possible. In my case, I got it done
within 1 minute to harvest an organ.
b. snap freeze tissue in a 1.5 mL tube in liquid nitrogen right after
harvesting. I dropped all tubes and left them there until I finished. I took
them out and stored them in -80 C freezer for RNA extration later.
c. if dissection is needed, do it in a 60 mm dish with prechilled buffer (I
prefer PBS) or medium (without serum) in ice bucket as quickly as possible
under a dissection micorscope. I did it within one minute or two minutes to
peel off the innermost layer of epithelial cells (mucosa).
Use a new dish for a new tissue. After dissection, put your tissue in 1.5
mL tube to snap freeze in liquid nitrogen.
d. Now it's the critical part. On the day of RNA extraction, take your
tissue samples out from -80C freezer and leave them in tubes in dry ice to
keep them frozen.
(1). Get a large styrofoam tray (rectangular shape) to be filled with dry
ice and to accommodate a metal plate.
(2). Prechill a metal plate (with or without hole for 1.5 mL tube) in a dry
ice box (container): just drop a metal plate in a dry ice box. You'll hear
some noise made by metal plate upon contacting the dry ice.
(3). Prechill a hammer in the same dry ice box. I bought my hammer at Home
Depot. better buy a relatively heavy hammer with a long handle.
(4). You have to have a special type plastic bag that can withstand freezing
and physical hammering as well as chemical (in this case, Trizol). In other
word, you don't want to have a leaky bag to hold your tissue samples for
RNA extraction. You also need a sealing tool (plug up to heat it up. It's
the tool that people use to make hybridization bags) to seal that plastic
bag to make smaller bags.You have to cut one side open if all four sides are
sealed by heating. In order to open easily the bag when it's chilled, I
always cut a notch along the open edge using sissors. Prechill these small
bags on dry ice in the tray.
(5). Take that prechilled metal plate and put it into the tray. Add more dry
ice into the tray and put some dry pellets on top of the metal plate to
keep it frozen.
(6). Prechill a clean forceps (use lavish amount RNAZap to clean your
forceps, pipettor, and your hands, every once a while, or whenever RNase
contamination is in question) in that tray with dry ice. Use forceps to pick
up your frozen tissue samples, if you accidently drop your sample into dry
ice tray instead of bag.
(7). Take your tissue samples out and bury them in dry ice in the tray.
(8). Get a lab helper now to help add Trizol for you when it's needed.
(9). clean out a small flat area on the metal plate without dry ice pellets.
(10). Get a 1.5 mL tube with your tissue sample, lay the tube upside down (
tip up, lid touches the metal plate flatly and evenly). Use the hammer to
gently strike the tip of the tube to dislodge your tissue. You'll get the
tissue free for sure. Don't worry if you crack the tube a little bit.
(11). Ask your lab helper (co-worker) to open the bag, but keep it open on
the metal plate or at least to put the bag back on metal plate after you
drop the
tissue into the bag. Open the tube and drop the tissue into the bag. Try to
drop the tissue to a corner of the bag. If not in the corner, try to bulge
the bag and gently push the tissue to the corner. If it's not in the corner,
it's still OK.
(12). Put the bag with tissue on metal plate. Put some dry ice pellets on
the top the bag for a while to keep tissue frozen.
(13). fold the bag to as small as possible and hold the open side tightly
for a seal.
(14). Hammer the tissue in your bag gently for a few times to make small
chunks. If you hammer violently, you'll send the chunks all over the place
inside the bag by explosion.
(15). keep hammering to make fine powder of your tissue.
(16). Try to collect them to a corner by buldging and tapping your bag
gently. Make sure the seal is there all the time.
(17). You unfold and open the bag. Ask your helper to add Trizol (say, 1 mL
ice-cold Trizol) into the bag with many quick triturations (pipetting ups
and downs quickly)to rinse all powdered tissue particles into Trizol.
(18). Suck the Trizoled tissue powder into a 1.5 mL tube and vortexed for a
while and let it sit on a tube rack for 5 minutes at room temperature with
occasional vortexing,then put it on ice. You may still see fine particle in
the Trizol. It's OK.
(19). RNAEasy to extract total RNAs.
The whole process may sound difficult and time-consuming, but it's the
fastest method to me, after you get used to it.
The key to a successful RNA extraction: harvest tissue as quickly as
possible; keep tissue frozen all the time.
RNA integrity (quality): RIN (RNA Integrity Number) by Agilent or RNA
Quality Index (RQI) by Bio-Rad is near 10 or 10 for all total RNAs.These
RNAs are perfect for microarray. Although partially degraded RNAs (like RIN
=6) can be used for microarray, it will be more advantageous (more
authentic representation of your tissue samples) to have perfect RNAs
for microarray.
The other advantage of this method: You can use this method to extract total
protein easily and quickly from tissue samples. Do the same smashing, but
add lysis buffer (not Trizol) in the final step. It worked perfectly for me
to extract protein from my tiny tissue samples.
I cannot remember the name of the plastic bag and its vendor. If interested
and want to know more in detail, please send an email to me (站内邮箱). I'
ll find it out later.
If it can be of any help to you, 伪币 is welcome.
Thanks.
avatar
n*k
17
不顶没天理呀!另外,你肯定是个好CO_WORKER!
不过,真的用的这样吗?我以前用了很多RNALATER提组织,从来没有出过什么问题,不
过我没用检查过RIN,也不是做去基因组的东西,你说的让我都想现在去提提试试,查
一下RIN;有没有谁用过RNALATER得到很好的RIN的冒个泡,或者是与LZ一样的,我有点
不敢相信的说

RIN

【在 M*****e 的大作中提到】
: 我的办法可能能帮你解决问题.
: 我曾经试过多种办法提没有降解(用Bio-Rad or Agilent Bioanalyzer测integrity or
: quality (RNA Integrity Number (RIN) by Agilent or RNA Quality Index (RQI)
: by Bio-Rad),不是仅仅用Nanodrop测RNA integrity or quality)的total RNA,然后
: 做microarray.
: 1.液氮研磨:RNA quality好,可以做microarray,但是麻烦和太慢(原因你应该知道
: )。
: 2. Glass tissue homogenizer: it didn't work for me to grind tissue in
: Trizol in ice bucket with ice, although my tissue was thin and tiny.
: Bioanalyzer showed RNA degradation.It's also low through-put.

avatar
c*1
18
好贴留名。
顺便问问,有用RNAlater dissection sample,然后做western的么?
我试过,好像没有问题。
avatar
n*k
19
In principle, RNAlater will pretty much inhibit everything, so it shall be
good for protein, RNA and DNA...but I never tried on protein or DNA...

【在 c****1 的大作中提到】
: 好贴留名。
: 顺便问问,有用RNAlater dissection sample,然后做western的么?
: 我试过,好像没有问题。

avatar
b*n
20
prostate这个组织确实很tricky
因为这玩意本身有很多腺体,在解剖的时候会释放出大量的各种RNase protease之类的
东西,所以只用TRIZOL的话RNA会降解
所以要么就用RNA later,可以保证不降解
要么就是液氮低温处理再加TRIZOL
你最好问问专门做prostate的人要他们的protocol看看,可以少走些弯路

【在 v****e 的大作中提到】
: 最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
: 不幸。
: dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
: LATER里比较难剥,因为变硬了。
: 然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
: 最后用RNAEASY纯化,
: 大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢

avatar
s*u
21
不同的20个老鼠的RNA 不要混合 for microarray.

【在 v****e 的大作中提到】
: 最近用老鼠组织提RNA,准备做microarray,可是RNA降解了。损失了`20个老鼠,非常
: 不幸。
: dissect tissue,每个老鼠take 10分钟,因为比较难剥。是不是这个原因?放在RNA
: LATER里比较难剥,因为变硬了。
: 然后就直接把tiisue放在液氮里了。抽的时候,放在trizol里。用匀浆器打散,抽提。
: 最后用RNAEASY纯化,
: 大家觉得有什么问题?有人和我说用液氮磨碎组织再加trizol,有用吗?谢谢

avatar
l*1
22
[回复] [回信给作者] [本篇全文] [本讨论区] [修改] [删除] [转寄] [转贴] [收藏]
[举报]

0

En 等 ManDate也去魔都开个千人级别以上的lab的时侯 两个可以好好切磋下RNA
later to be or not to be?
[ 14 ]
发信人: neverthink (nevernetbug), 信区: Biology
标 题: Re: 我的办法可能能帮你解决问题. 我反对用RNALater。
发信站: BBS 未名空间站 (Sat Jun 16 17:52:42 2012, 美东)
不顶没天理呀!另外,你肯定是个好CO_WORKER!
不过,真的用的这样吗?我以前用了很多RNALATER提组织,从来没有出过什么问题,不
过我没用检查过RIN,也不是做去基因组的东西,你说的让我都想现在去提提试试,查
一下RIN;有没有谁用过RNALATER得到很好的RIN的冒个泡,或者是与LZ一样的,我有点
不敢相信的说

RIN

【在 M*****e 的大作中提到】
: 我的办法可能能帮你解决问题.
: 我曾经试过多种办法提没有降解(用Bio-Rad or Agilent Bioanalyzer测integrity or
: quality (RNA Integrity Number (RIN) by Agilent or RNA Quality Index (RQI)
: by Bio-Rad),不是仅仅用Nanodrop测RNA integrity or quality)的total RNA,然后
: 做microarray.
: 1.液氮研磨:RNA quality好,可以做microarray,但是麻烦和太慢(原因你应该知道
: )。
: 2. Glass tissue homogenizer: it didn't work for me to grind tissue in
: Trizol in ice bucket with ice, although my tissue was thin and tiny.
: Bioanalyzer showed RNA degradation.It's also low through-put.

avatar
u*d
23
赞!


or

【在 M*****e 的大作中提到】
: 我的办法可能能帮你解决问题.
: 我曾经试过多种办法提没有降解(用Bio-Rad or Agilent Bioanalyzer测integrity or
: quality (RNA Integrity Number (RIN) by Agilent or RNA Quality Index (RQI)
: by Bio-Rad),不是仅仅用Nanodrop测RNA integrity or quality)的total RNA,然后
: 做microarray.
: 1.液氮研磨:RNA quality好,可以做microarray,但是麻烦和太慢(原因你应该知道
: )。
: 2. Glass tissue homogenizer: it didn't work for me to grind tissue in
: Trizol in ice bucket with ice, although my tissue was thin and tiny.
: Bioanalyzer showed RNA degradation.It's also low through-put.

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