|
M**a
发帖数: 4816 | 2 try NuGEN RNA amplification kits.
I even got high quality cDNA good for NGS from a single cell (human or
bacteria).
Array? Why not NGS? |
|
j*p
发帖数: 411 | 3 Try RNAseq ba. As far as I know, RNAseq requires less starting material, as
few as 100 cells, based on my knowledge (I have never seen RNAseq data from
single cell).
Besides, if you decide to use RNAseq, I suggest NuGEN RNA amplification kits
, because data from NuGEN is the best I have ever see, in comparing to at
least 3 other kits. |
|
r*m
发帖数: 16380 | 4 这是为什么呢？
QPCR用两个primers， 各20-25bp，nanoString也是两个probe， 各50bp，而且没有
amplification，至少从表面上看不出来QPCR更准确啊。
more |
|
t***u
发帖数: 119 | 5 qPCR, co-amplification of the target and reference genes in the same
reaction well. ABI has predesigned primers for lots of genes CNV |
|
l**********1
发帖数: 5204 | 6 RE LZ:
pls try
Cassette-ligation downstream 3'RACE PCR
or Tail-PCR
Protocol:
看图说话
>
Amplification of FSTs by 3’-RACE
Total RNA was isolated using hot-phenol extraction
[27], from 20 ml cultures in 50 ml Falcon tubes on a rotating
wheel. Reverse transcription used the SuperScript
III First-Strand kit from Invitrogen in a DNA Engine
PCR machine (MJ research). Primer QT was mixed with
5 μg RNA, incubated at 65 °C for 5 min then cooled to
55 °C over 100 sec, after which the annealed mixture
was kept... 阅读全帖 |
|
m****e
发帖数: 489 | 7 Nucleosides, Nucleotides and Nucleic Acids
Volume 27, Issue 3, 2008
Nucleic Acid Isothermal Amplification Technologies—A Review
Can you please send to a********[email protected]? Many thanks!!! |
|
m****e
发帖数: 489 | 8 Rapid DNA detection by beacon-assisted detection amplification
Ashley R Connolly1 & Matt Trau1
AffiliationsContributionsCorresponding author Journal name:
Nature Protocols
Volume:
6,
Pages:
772–778
Year published:
(2011)
DOI:
doi:10.1038/nprot.2011.326
Published online12 May 2011
Please send to a********[email protected]. Many thanks!! |
|
l**********1
发帖数: 5204 | 9 Sounds reasonable.
But key point is 让人怀疑作者检测到的human HBV infected cccDNA是否是真的
human HBV infected cccDNA?
details please refer
. 文章中用Southern blotting检测到了cccDNA的形成。cccDNA是HBV转录的膜板，被认
为是HBV持续感染的关键因素。从结果来看cccDNA的形成时间与HBV蛋白、RNA和DNA相比
比较早，尤其量很高。其实对于HBV来说哪怕是CMV启动的HBV（比如pCMV-HBV）cccDNA
的量都是很低的，更不要说是自身启动子启动的了。在NTCP介导的HBV复制水平整体很
低的情况下，具有高水平的cccDNA形成，让人怀疑作者检测到的cccDNA是否是真的
cccDNA，所以作者必须证明他们所指示的条带是真的是cccDNA，必须用EcoRI酶切（
cccDNA能被EcoRI线性化）和加热煮沸cccDNA（沸腾后不会被解成单链）来确证，同时
必须要有阳性对照。当然对于鸭乙肝病毒（DHBV）
http://blog.scien... 阅读全帖 |
|
l**********1
发帖数: 5204 | 10 Sounds reasonable.
But key point is 让人怀疑作者检测到的human HBV infected cccDNA是否是真的
human HBV infected cccDNA?
details please refer
. 文章中用Southern blotting检测到了cccDNA的形成。cccDNA是HBV转录的膜板，被认
为是HBV持续感染的关键因素。从结果来看cccDNA的形成时间与HBV蛋白、RNA和DNA相比
比较早，尤其量很高。其实对于HBV来说哪怕是CMV启动的HBV（比如pCMV-HBV）cccDNA
的量都是很低的，更不要说是自身启动子启动的了。在NTCP介导的HBV复制水平整体很
低的情况下，具有高水平的cccDNA形成，让人怀疑作者检测到的cccDNA是否是真的
cccDNA，所以作者必须证明他们所指示的条带是真的是cccDNA，必须用EcoRI酶切（
cccDNA能被EcoRI线性化）和加热煮沸cccDNA（沸腾后不会被解成单链）来确证，同时
必须要有阳性对照。当然对于鸭乙肝病毒（DHBV）
http://blog.scien... 阅读全帖 |
|
f****e
发帖数: 679 | 11 2nd this. The amplification efficiency of Taq drops sharply when the
amplified fragment is more than 6-7 kb. Phusion works ok for 10 kb, plus it
is fast, saves a lot of time. |
|
q**********0
发帖数: 335 | 12 Thanks for the above sharing. Not sure how high the incorporate error rate
of phusion or PfuUltra high-fidelity DNA polymerase. Do both of them have
high-fidelity amplification? Thanks. |
|
s******9
发帖数: 283 | 13 In principal, long template should affect the cluster density. Weird
clusters mean non-circular ones. Standard clusters usually have a round
shape.
Somebody told me 1000bp worked for him, which tells the robustness of this
amplification method. But I guess 1000bp can compromise your data quality. |
|
a******a
发帖数: 283 | 14 I have one question that's really baffling me. I have a set of primers, only
gave good efficiency and good amplification signals when primer
concentrations at 2.5uM (working concentration!!! triple checked), annealing
temperature at 63.8C (while the Tm for the primers are 54.6C and 55.8C).
Anyone can explain at all? |
|
G*****h
发帖数: 320 | 15 如果primers 的特异性很好，没有杂带，没有intons，那么很可能就是target gene 本
身的拷贝数就很低。
如果物种的 genome 已经测序过的，可以看看这个基因的拷贝数。也可以算一下一个细
胞的 DNA 大概是多少 DNA，然后算算你放进去的DNA的量可以折算成几个拷贝。
一般1个bp 大概 10^(-9) pg。1 Mb gDNA ～1 fg。如果一套基因组大小是 1 GB，那么
～1000 fg ＝ ～1 pg。如果你放了 10 ng，单拷贝的基因就是 10^4 拷贝。
当然 genome PCR 的条件也需要 optimized。比如先要 95 C denature 5 min，前面的
一些 cycles 的annealing 和 extension 的时间长些，等等。
再就是根据你 amplify plasmid的结果（slope），看看你的 PCR amplification 的
efficiency。如果 efficiency 很低，那么大genome 里面的低拷贝的基因就更需要多
个 cycles。 |
|
l*******i
发帖数: 153 | 16 qPCR测了一批基因在不同时间点的表达（1-6天），现在作图时碰到一个问题：
因为所有时间点、所测基因的表达都要与未处理组(即day 0)比较，所以我在计算时，
把每个基因在未处理组中的表达设为 1， 这样计算不同时间点基因变化的倍数。其中
有两个基因在未处理组不表达（program给出的CT值为undetermined,即未
amplification）,这样计算时就无法得到dela CT，也就没法作图。
我的想法是把没有扩增的基因的CT值设为最大CT值，即40，然后计算delta CT，以方便
作图。但是，实验室博后认为这样不对，是artificial effect；正确的做法，应该用
positive control，然后未处理组和各个时间点的都与positive control比较。我觉得
这样很混乱，因为是一批基因，按博后的意见的话，我得每一个基因对应一个positive
control,做出的图不太好理解。
不知各位有无碰到这种情况？你们都是怎么处理的呢？最好能提供一两篇有关类似问题
的文献。谢谢！ |
|
D********0
发帖数: 409 | 17 1. dilute template，
2. 如果不work，把引物往里设计，primer binding的时候不能perfect match到原来引
物位置。
3. 如果不purify PCR product，里面焦磷酸盐之类的会降低PCR specificity and
sensitivity。
4. qiagen hotstar kit for amplification。 |
|
C*******e
发帖数: 4348 | 18 模版是什么？
cDNA？
细菌gDNA?
人／老鼠gDNA?
用过takara的LA(long and accurate)
扩～12kb，细菌gDNA
然后TOPO blunt end
测序，20个里面拿到一个全长都正确的
不过这是几年前
现在不知道哪一家long amplification做得最好
虽然每家都说自己的最好LOL |
|
G*****h
发帖数: 320 | 19 Design several pairs of primers and then run BLAST searches of these primers
against the host genome sequences (and closely related species) if they are
available.
Rule out these primers with high identities, particularly those with
relatively long identical sequences at 3' ends.
Make sure that 3' ends of your primers have at least one base (the more, the
better) that does not match any host genome sequences.
Test your primers using host genome DNA alone to ensure there are no non-
specific ampl... 阅读全帖 |
|
N****n
发帖数: 294 | 20 Does anyone have the paper describing MALBAC（multiple annealing and looping
-based amplification cycles) ? Appreciate it if you can send a copy.
Science 21 December 2012:
Vol. 338 no. 6114 pp. 1622-1626
DOI: 10.1126/science.1229164
REPORT
Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a
Single Human Cell
Chenghang Zong1,*, Sijia Lu1,*,†, Alec R. Chapman1,2,*, X. Sunney Xie1
,‡ |
|
d****7
发帖数: 109 | 21 check this paper from Rick Young's lab, it might help: Transcriptional
Amplification in Tumor Cells with Elevated c-Myc |
|
A****w
发帖数: 244 | 22 最近在做baculovirus transfection一直做不出来
用了两种不同的方法，一个是BD BaculoGold DNA ＋ pVL1393-DNA.
就是follow manual
另一是Bac to Bac system， 用cellfectin II reagent。
用的是Sf-21 conditioned in Sf-900II media （大于95％viability)
怎么做都在5天transfection后看不到病态细胞
然后amplification后也没有～
请大牛指教！
感谢！ |
|
n***3
发帖数: 663 | 23 是不是根本就没有拿到recombinant baculovirus啊？我们做的时候7天以后Sf9 cells
就好多死细胞了。Sf21应该也一样啊。
“amplification后也没有”是什么意思？没有检测到目标蛋白基因的表达？ |
|
A****w
发帖数: 244 | 24 BD BaculoGold DNA ＋ pVL1393-DNA 那个没有bacmid prep的这一步
bac to bac BACMID DNA用M13和目标基因的特异引物检测的，可以看到pcr产物，难道
是transfection的量不够，用quick and dirty prep的。
》》是不是根本就没有拿到recombinant baculovirus啊？我们做的时候7天以后Sf9
cells
就好多死细胞了。Sf21应该也一样啊。
感觉是没有拿到baculovirus，细胞transfection 5－7天后，看不到涨大的病态细胞（
或者和control差别不大）看不出来。。。
有些时候看到是好多死细胞了，有的时候看起来还行。
》》“amplification后也没有”是什么意思？没有检测到目标蛋白基因的表达？
就是在transfection后，有这个supernatant去infect健康的细胞（在T75里）。然后看
细胞形态，也做蛋白表达检测（western） |
|
r***e
发帖数: 2539 | 25 不是说myc没有具体的target吗？是整个转录系统放大。
看最近的两篇back to back文章在Cell。
Lin, C. Y. et al. Transcriptional amplification in tumor cells with elev
ated c-Myc. Cell 151, 56–67 (2012)
Nie, Z. et al. c-Myc is a universal amplifier of expressed genes in lymp
hocytes and embryonic stem cells. Cell 151, 68–79 (2012)
http://www.nature.com/nrg/journal/v13/n11/full/nrg3364.html |
|
l**********1
发帖数: 5204 | 26 BMJ
Gut is not trash journal
this journal 2013 one review
web link:
http://gut.bmj.com/content/62/8/1093.extract
its pp1094 left column top
cited here stuff sentence,
>Surprisingly, Yan et al did not provide
>Southern blot data demonstrating accumulation
of HBV DNA replication intermediates,
as this electrophoretic pattern is
a signature of productive virus replication.
Only one subfigure shows some evidence
of cccDNA formation, which
defines a successful infection by HBV;
however, unlike HBV DN... 阅读全帖 |
|
a*****t
发帖数: 81 | 27 strictly speaking, CNV refers regions of amplification or deletion larger
than 1kb. |
|
l********n
发帖数: 260 | 28 揭露臧敬五在新闻媒体上的谎言——兼评其JCI论文
作者：胆大妄为
方舟子先生，
你好！本人注意到臧敬五在很多新闻媒体上对其作为通讯作者发表在JCI上
的论文加以宣传后，翻阅了这篇论文（1），发现臧敬五胆大妄为，编造谎言，
欺骗中国新闻媒体，应予以揭露。
先看二篇臧敬五在媒体上的宣传：
“骨桥蛋白”是引发类风湿性关节炎的顽凶
新华网上海３月２日电（胡德荣、仇逸）严重影响人类健康和生活质量的类
风湿性关节炎（注：rheumatoid arthritis，RA）研究获得重大突破性进展。我
国科研人员在国际上第一次阐明了骨桥蛋白（注：osteopontin ，OPN）在类风
湿性关节炎发病中的作用，发现“骨桥蛋白”是引发类风湿性关节炎的顽凶，为
治疗类风湿性关节炎找到了临床手段和药物靶点。……
http://www.stcsm.gov.cn/news/detail.asp?pid=64857
我专家突破类风湿关节炎治疗世界难题
上海分院
日前，中国科学院上海生命科学研究院健康科学中心主任臧敬五教授，带领
科研人员完成了“骨桥蛋白在类风湿性关节炎中的病理机制”研究，其论文已刊
登在3月2日出版的... 阅读全帖 |
|
y****6
发帖数: 196 | 29 It may be a little misleading. But ACD does call the intermediate probes "
preamplifier" and "amplifier" because they allow signal amplification. I am
interested in this technology, but have not tried it.
Are
: your refering to pre-hyb?
: The overall procedure is similar to regular in situ, but you should
follow
: their manual to optimize your experiments. |
|
s******9
发帖数: 283 | 30 When you deal with less than 100 copies, you need to consider RT-PCR as a
series of random processes (e.g., Poisson): 1) template destruction, which
is significant for RNA templates, 2) distribution of successful cDNA
synthesis, and 3) distribution of successful cDNA amplification in each
cycle. Many stochastic variations can affect a final output. |
|
X***n
发帖数: 366 | 31 brilliant method.
locus-specific probe coupled with rolling circle amplification. |
|
l******u
发帖数: 936 | 32 the technology is original from Ulf Landegren lab from Uppsala University,
Sweden. Ulf is a member of the royal swedish academy of sciences, he
invented Pad Lock Probe and Proximity ligation assay. The technologies in
the paper are based on Proximity Ligation assay + RCA (rolling circle
amplification).
in |
|
l******u
发帖数: 936 | 33 depend on what are you doing.
what you talking about is the bio-marker test for some target therapy drug,
but actually, the precis therapy ( personalized therapy ) using chemo-drug
or cocktail drug (combining different chemo-drug or chemo/target drug) might
be based on genomic mutation profile using PDX animal model test and bio-
big data on big PDX/patient cohort. This is the real challenging topic for
precis therapy.
you can quantify the DNA amplification/deletion using ArrayCGH and you
can al... 阅读全帖 |
|
D***s
发帖数: 5613 | 34 用过invitrogen(Life technology)的5' RACE System for Rapid Amplification of
cDNA Ends, Version 2.0，感觉还可以，GSP引物设计比较重要，最好多设计几条 |
|
D**R
发帖数: 371 | 35 最近做了一批RNA Extraciton，用NanoDrop测时碰到一个问题，大部分的RNA value都
在范围之内5-25 ng/ul，可是有两三个的samples值很高，超过200ng/ul，但是qPCR却
一点amplification也没有，那那个OD260 peak到底是什么东西呢？我们的是人的
sample，那些有bacterial contamination？怎么可以把它们去掉呢？多谢多谢 |
|
D**R
发帖数: 371 | 36 多谢楼上两位，我倒也不是追究NanoDrop的准确性，关键是有两个(2/30)samples给了
两个实在是非常漂亮的peak at OD260，但是qPCR啥也没有，那种低得都测不准的
samples反倒是出来amplification了，所以我很困惑那么高的band到底是什么东西，
bioanalyzer也ran一下，觉得可能有些gDNA和RNA降解了，但关键这一批samples来自同
一个site，ralative speaking，这么高的peak会是从哪里来的。 |
|
D**R
发帖数: 371 | 37 我做的是qRT-PCR，那些测出来NanoDrop很低值的sample都有amplification出来，唯独
这几个看起来Peak很漂亮，OD260值很高的啥也没有，所以我很困惑那都是些什么东西
，maybe contaminated with bacterial RNA? |
|
D**R
发帖数: 371 | 38 SurePrep RNA extraction kit from fisher, it's using column, no phenol
chloroform.
Actually I have really nice peak at 260, but no RT-PCR amplification. |
|
d********6
发帖数: 76 | 39 这种kit里边肯定都是有phenol的，不管是什么，这个东西抑制了你下一步的反应，而
且我感觉应该不是DNA。你可以直接用这个sample做PCR（不做RT），如果有DNA肯定会
有amplification的。我感觉这个PCR应该什么都没有，因为你的sample里边会有东西抑
制酶反应。 |
|
m******g
发帖数: 467 | 40 Science 13.11.15 (包含Editor's Choice等推荐的其他杂志的文章，之后省略这句话)
推荐：
1. Dosage compensation via transposable element mediated rewiring of a
regulatory network
D. miranda在1 my前出现neo-X chromosome, TE-mediated rewiring of regulatory
network (domestication, amplification, and fine-tuning) followed by erosion
of nonfunctional parts of the transposon
idea挺有趣的.
2. Long-term dynamics of adaptation in asexual populations
Power law model fits/predicts the data better than Hyperbolic model with an
asymptote.
Fitne... 阅读全帖 |
|
m******g
发帖数: 467 | 41 Science 13.11.15 (包含Editor's Choice等推荐的其他杂志的文章，之后省略这句话)
推荐：
1. Dosage compensation via transposable element mediated rewiring of a
regulatory network
D. miranda在1 my前出现neo-X chromosome, TE-mediated rewiring of regulatory
network (domestication, amplification, and fine-tuning) followed by erosion
of nonfunctional parts of the transposon
idea挺有趣的.
2. Long-term dynamics of adaptation in asexual populations
Power law model fits/predicts the data better than Hyperbolic model with an
asymptote.
Fitne... 阅读全帖 |
|
p**********t
发帖数: 2636 | 42 ExTaq is great for PCR amplification from low consentration or highly
degraded DNA template. It is much more sensitive than many other taq that I
tried.
My former lab ruitinely deals with decades' old plant samples with lots of
secondary compounds. We tried many diffent taq and settled on ExTaq
No idea about the connection between exonuclease and sensitivities. |
|
Z******5
发帖数: 435 | 43 用invitrogen的这个kit
5' RACE System for Rapid Amplification of cDNA Ends, version 2.0
说明书上说貌似50min反转录4kb没问题。 我之前做大约10kb的，反转录2个小时，结果
很好。 不过我那个基因表达丰度非常高。如果表达丰度很低的，不知道效果会如何。
另外，建议在预测的TSS后面1-2kb的地方同时设计一个引物做5'RACE， 看看和你从
11kb的位置拉出来的是不是同一个起始位点。 |
|
o*******a
发帖数: 242 | 44 I think un-differentiation is not a basic characteristic of cancer. There
are many types of normal cells that are not differentiated at all, such as
progenitor cells or stem cells, but, they are not cancerous. Also, certain
cancers are sometimes well differentiated. But, cancer cells must have the
ability to proliferate unlimitedly or immortalize. Ki67 or other markers
may not be good methods to assess the proliferation in some cancers.
Pathological assessment in differentiation is a good indica... 阅读全帖 |
|
|
z*******6
发帖数: 679 | 46 号称可以RNA amplification的吧？... |
|
l***y
发帖数: 638 | 47 理论上讲Taqman的特异性要高过sybr green，因为它只有在probe结合在PCR扩增的区间
才会有信号，PCR的non-specific amplification或者probe的non-specific binding两
者之一的情况都不会产生信号，而sybr green只要有非特异PCR扩增就会出信号
另外Taqman可以做internal control，一个孔同时测未知基因和housekeeping，可以降
低部分variation。
Taqman就是太贵了，一般做做实验sybr green也就差不多了，反正qPCR也就是一个data
，真要做solid肯定得有其他independent的方法验证。
fluorescent |
|
B******o
发帖数: 496 | 48 probe's non-specific binding can generate signal as well, as long as the
amplified DNA has similar sequence that can hybridize with the probe. It
really depends on how the probe is designed.
As for Sybr Green assay, it is necessary to check melting curve every time.
If any non-specific amplification were present, the curve wont be a single
peak.
data |
|
z*******6
发帖数: 679 | 49 大家有人用HMS的那个qPCR primer bank吗？里面有很多都validate过的... 我感觉很
好用...
看了几位大牛的回复，确实到不少东西... 但技术层面上讲，我也差不多是相信32以下
的，然后再用几次独立实验reproduce... 我的ct一般在0.3到0.5以内... 用的是
repeater和排枪的组合...
嗯，看了大家的回复，我也在想我要不要做一个amplification efficiency的test和
linearity的test... |
|
a*******a
发帖数: 4233 | 50 http://news.sciencemag.org/asiapacific/2014/09/exclusive-nature
EXCLUSIVE: Nature reviewers not persuaded by initial STAP stem cell papers
As two discredited, and now retracted, stem cell papers have produced an
almost unimaginable fallout—a national hero accused of scientific fraud,
the revamping of one of Japan’s major research institutes, and the suicide
of a respected cell biologist—researchers have privately and publicly asked
how Nature could have published work that, in retrospect, seems ... 阅读全帖 |
|
