j******b
发帖数: 78 | 1 you may consider to flush the bone (tibia or femur) with 1ml Trizol directly
. should be able to get enough RNA for RT, Realtime etc.
BTW, freeze shouldn't affect your RNA yield.Just make sure the samples are
at room temperatur when you start Trizol isolation. |
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m****a
发帖数: 427 | 2 用kit提了RNA,浓度还可以但是260/280=2.2了，这样是不是说RNA已经降解了呀，还能
用吗？还有一个问题，在提取的时候匀浆那一步是不是要在冰上进行？谢谢啦 |
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a***e
发帖数: 1010 | 3 No, it does matter.
The contamination of DNA in the RNA sample will mask the fold change
caused by RNAi.
For example, your RNA is 10 before treatment, but 1 after treatment. Thus
the fold change is 10.
However, if you have 5 part of DNA in your sample, the fold change 15/6 =
2.5 fold.
DNAase |
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f*********r
发帖数: 1233 | 4 不用这么小心翼翼。
如果WB显示KD了50-80%，RNA应该没啥问题。跑一个RT-PCR看看就是了。如果KD水平没
有达到90%以上，再去考虑加DNase。
如果WB显示KD水平小于50%，甚至不足1/3，就不用琢磨RNA了。 |
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a***e
发帖数: 1010 | 5 I agree that it may be not that important to treat RNA sample with DNaseI
during the RNA purification process in the original question. However, it is
better to maintain a "good habit" to do so.
There are several reasons for this:
(1) it doesn't add many extra work. DNaseI treatment is very simple and fast
. Qiagen has on-column DNaseI digestion kit. Or an extra TRIzol purification
step only adds one more hour.
(2) if the experiments include transfection of plasmid to overexpress some
genes or s |
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H*****e
发帖数: 120 | 6 Hi,
I have some samples that I prepared for microarray. When I went to the core
, I noticed that the core has only a few people do array now but most of
them do RNA-seq. They pass me some information like I know everything. But
, after read them for the whole morning, I still no idea what to do.
Especially,
1. Library: What is the difference and advantage between Single Read and
Paired end and mRNA and small RNA libraries.
2. Lane: some run one lane and some run 1/2 lane.
3. Cycle numbers: h |
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s******y
发帖数: 28562 | 7 NO. Trypsin is a protease and won't bother with RNA.
Also, in most RNA extraction kit, there will be a step to denature all the
protein, that includes protease. |
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y***j
发帖数: 11235 | 8 做一个实验， 细胞需要染色然后sort一下再提RNA。
染色至少冰上30min，另外再flow cytometer里有几十秒钟的时间温度会升到RT甚至更
高.会不会有影响呢？
formalin fix一下再染色会不会影响下一步的RNA提取呢？准备用qiagen的RNeasy
micro kit.
或者有什么其他专门的可以再进细胞的RNAse inhibitor么？ |
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l******u
发帖数: 936 | 9 你fix, 然后还有染色RNA 还要 facs
RNA都降解的很多了, RIN number 很难到6.0
做array 就不可靠了. |
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w******e
发帖数: 1187 | 10 土人第一次做，全盘follow trizol的protocol。为省reagent用了100mg tissue
per ml trizol。发现以下问题，请达人指点：
1. chloroform extraction后的precipitation，为什么用isopropanol？室温
incubate也让我很困惑。。。
2. 做了两次，第一次是heart，拿到RNA很顺利，测OD时发现：260 peak偏向270；
230附近有极高的峰。看来chloroform和precipitate后wash的步骤都不太effective？
3. 第二次做liver，搞到巨多RNA，很oily，dry了之后pellet死活不溶，试了
60oC加热，加大volume，最后还是有很多不溶。是不是trizol用少了？
4. 用了hand held homogenizer和pestle+mortar两种tissue homogenization
methods，run了urea agarose gel（顺便请问大家都用什么denaturing gel），
18S:28S rRNA大约1：1，很困惑degra |
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O*********e
发帖数: 79 | 11 Those are total RNA isolated from macrophages. I used TE buffer to try to
dissolve the RNA pellets. But the pellets don't go to solution even after I
tried to heat them up to 56 degree. Does anybody have a better idea? Thanks
a lot! |
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w***a
发帖数: 4361 | 12 Macrophages seem to be different from other cell types. I have exactly
the same experience before. Eventully, I switched to Qiagen RNeasy Kit
for RNA extraction. The yield is lower, while the RNA quality is good enough. |
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l*****c
发帖数: 17 | 13 你的描述太少，
（1）1000多个RNA-Seq reads，有些难以想象，RNA-seq都是以M来计算的啊，是1000M
Reads？
（2）是测序的有参考基因组序列的，比如human还是完全新的？有很多软件完成不同的
任务，比如abyss,soapdenovo,velvet,cufflinks,scripture等等。 |
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m****M
发帖数: 360 | 14 Of course not. I got perfect ratios of my RNA samples from nanodrop, but when
I checked them on bioanalyser, they were all degraded (around 7 or lower).
A simple example, you can get perfect ratio when you measure DNA primers. I
have done a lot RNA work, trust me or not? It depends on yourself. -:) |
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q**********0
发帖数: 335 | 15 Recently, I tried twice by using MEGAscript kit-making RNA Probes (32P-label
GTP and incorporated) in sourthern Blot. However, all failed. The probe is
single-stranded RNA, it should blot the DNA template. I don't know what's
wrong with it. Anybody have the experience on this? Please help. Thanks. |
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c******r
发帖数: 3778 | 16 what marker did you use? and what marker did they use?
if you used DNA marker which were double stranded, your single strand RNA should run faster than the marker of the same sized DNA.
so my guess is you used DNA markers, but they used RNA markers. |
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m***n
发帖数: 536 | 17 现在我们想检测转染细胞后到底有多少exogenous micro RNA和protein of interest结
合（比较在不同条件下结合量的差别）。
请问有谁做过这个？micro RNA很短，只有20多个碱基，这个PCR的原理是什么？具体怎
么设计引物呢？
商业化的kit里都有设计好的引物甚至RT的引物。就是不太明白整个的原理，和传统PCR
的区别是什么？ |
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C*******e
发帖数: 4348 | 18 要看lz要做什么了
现在差不多每个出RNA kit的公司也都有专提或者enrich mRNA，small RNA的kit |
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n********k
发帖数: 2818 | 19 Hey, folks:
We are planning to perform some ChIP/RNA-Seq as well as miRNA array
analysis some time soon. I was wondering any of you could kind of share
some of your experience in general.
Thanks a lot.
Something like: which services do you use(in house, or company), software,
etc etc. Which protocols, Kits? Pros and Cons, critical steps and tricks...
how many reads do you have, is it enough to cover the genome or enough for
the purpose? replicates? what about mRNA v lncRNA v miRNA, do y... 阅读全帖 |
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c*******s
发帖数: 29 | 20 I think you should use the same proportion of the nuclear RNA and
cytoplasmic RNA for the RT-PCR. What's the purpose of your control? You can
do RT-PCR for an pre-mRNA (containing introns which should be exported) to
show your fractionation is clean. |
|
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z***q
发帖数: 907 | 22 对的，镁离子浓度过高会使谱图看起来很差，所以一般不会超过5mM
但是，请问“要是想要稳定RNA结构,在buffer里面至少需要10mM”这个是对于任何浓度
的RNA都适用的么？
谢谢！
5mM |
|
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w******e
发帖数: 1187 | 24 多谢！加个问题哈：我看了几篇paper都选择T4 DNA ligase instead of RNA ligase,
不知道用RNA ligase有没有什么concern？ |
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f*******e
发帖数: 628 | 25 不知道你要“测”什么，是你的 RNA 的长度加量的组成，还是 sequence, 还是什么别
的。你的微量 RNA 如果是个混合物，用 bioanalyzer 出来是 smear，基本上看不出什
么来，更别说定量。如果是单一成分，用 bioanalyzer 才有可能。而且好像最好别太
小，低于 bioanalyzer 的最低 marker，就不行了。我用过的好像是 50 bases？
如果就是想知道一个总的量 （pg/uL)，可以用染色的 dye, 然后用可以测荧光的
nanodrop 来定量。 |
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c****r
发帖数: 199 | 26 请大家推荐个RNA amplification kit. 做了好几次FACS，好不容易提到30ng的total
RNA, 做QPCR不够用。从前用过Nugen的wt-ovation，好是好，就是太贵了。大家还有什
么好推荐？ |
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T**********r
发帖数: 287 | 27 从组织里分离细胞，之后FACS，直接sort到RLT裂解液里（from Qiagen RNeasy plus
mini kit),
获得cell 数量约20000，之后用RNeasy kit提取RNA，获得的RNA送去gene-profiling,
总是说质量
不好。
求有经验的同学给些建议，感激不尽。 |
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m*********n
发帖数: 215 | 28 Collect到RNA－later里（room temperature）。然后spin down再提取RNA。 |
|
|
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n******7
发帖数: 12463 | 31 cool!
不知道现在还没有可能在某个角落找到纯rna生命形式 |
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M*****n
发帖数: 16729 | 32 用CLC作RNA=seq是不是一个专门的module?
俺们有CLC不过是做genome分析的，要作RNA-seq是不是还要买这个功能？ |
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m******5
发帖数: 1383 | 33 以前没提过这么少的组织里出来的RNA
样品保存在RNA later里了
请问如何处理这样情况的样品？ |
|
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m***o
发帖数: 272 | 35 当然定量了。现在觉得如果离心了，分行的细胞会多胞浆的RNA。不离心，就是细胞核
和胞浆的都很多。离心对不分化的细胞影响不大。这样即使定量，离心和不离心获得的
RNA就在分化和不分化比较时就会不一样。 |
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W*****o
发帖数: 1780 | 36 Hi thanks for help. Ship RNA to China.
Last time I used dry ice and it was very expensive. This time I am going to
keep RNA in 70% ethanol and ship with ice bag (2-8C). Is it OK? Or I have to
request another service for -20C in a special box but expensive too. |
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z*******o
发帖数: 1794 | 37 不长，我做过96小时的，还是处理。楼主是不是没有用液氮存样？RNA哪里那么容易降
解？
我做过多年的RNA提取，最多一次平行样品有36个，似乎只有一次降解过。 |
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g********0
发帖数: 6201 | 38 我当初测２6０／２8０时无论怎么努力改善提取，更换溶解RNA的buffer，值就是偏
低。后来我想了一个终级方法解决了这个问题，就是－－－－－－不测了，直接用２6
０算浓度就行了，反正我用这种RNA从来没出过问题。 |
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s********n
发帖数: 248 | 39 http://gtac.wustl.edu/services/sequencing/analysis-pipelines.ph
RNA-Seq
TopHat – align raw sequence reads to the reference genome. Tophat maps across splice junctions.
Cufflinks - assemble the transcripts, estimate their abundance and test for differential expression in RNA-Seq samples. Currently gene abundance is restricted to annotated genes and transcripts. |
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h*******o
发帖数: 4884 | 40 For Isolation RNA from brain tissues, you need to choose RNA kits that are
specific for fat-rich tissues, regular kits do not work well. |
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b******s
发帖数: 193 | 41 先 感谢 楼上的 回答
其实我的问题是 punch 后 选择的kit，用于提高RNA 质量，而不是RNA的数量 |
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i*********0
发帖数: 915 | 42 问题是,有DNA 污染的total RNA样品可不可以做RNA seq?
谢谢! |
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e***o
发帖数: 344 | 43 感谢各位的回复。我要把一个报告基因和一段RNA分别在AAV里表达，因为AAV容量太小
。所以想用一个promoter。用bidirectional 的promoter是个很好的idea，不知道有没
有在老鼠里用的比较成熟。加IRES和酶切都只能保证两个蛋白，我有一个要用RNA.
ribozyme好像有戏，但是不熟悉，不知大虾们能不能具体讲讲。
谢谢 |
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n********k
发帖数: 2818 | 44 completely dry, right? What could happen to it? IDT sends out the RNA oligo
this way all the time...I think it is pretty safe with pure organ solvent
or dry at RT for long time...otherwise, it would be problematic when we dry
the RNA at the end of Trizol protocol, right?
题, |
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C*******e
发帖数: 4348 | 45 应该问题不大。
从化学角度看，RNA其实比DNA更稳定（化学稳定）
很多人不喜欢做RNA主要是因为RNase
RNase到处都是，而且不易灭活
既然是定的样品
如果他们保证RNase free的话
完全没有问题 |
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A******y
发帖数: 2041 | 46 What are you talking about? Chemically,RNA is not stable because of the
hydroxy group (self hydrolysis)...in his/her situation the RNA is dry, so I
think it will still be good. |
|
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x******m
发帖数: 736 | 48 trizol
for RNA-seq. needs high integrity RNA |
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x******m
发帖数: 736 | 49 trizol
for RNA-seq. needs high integrity RNA |
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w******n
发帖数: 767 | 50 哈，我遇到过，提小鼠肝脏RNA,最后有一大坨白色的，溶不了。解决方法请按照TRIZOL
说明书严格操作。
1.裂解要充分，悬液要透明，看不到没溶的组织。
2.氯仿抽提离心时间要足够。
3.4度离心。
最后得到RNA应该是速溶的。 |
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