b****s
发帖数: 148 | 1 应该在differentiation时才表达的2个gene，最近一直在proliferation的时候就表达
了，重试
了很多遍都是这样。
开始以为是confluency太高，有部分differentiation，就连续几天控制在5%以下，再
取cfl 20%
时的RNA，还是发现gene表达了。
确定Growth Medium没问题，那2个gene也不是什么新发现，cell line也是实验室经常
用的，一筹
莫展。。。。。。 |
p******d
发帖数: 3737 | |
b****s
发帖数: 148 | 3 试过了，用新化的freeze back试过两次，还都是一样
【在 p******d 的大作中提到】
: 有原来冻存的细胞吗？从新化一只试试？
|
a***e
发帖数: 1010 | 4 应该在differentiation时才表达的2个gene???????????????????????
who said this? |
s******y
发帖数: 28562 | 5 一般基因的表达没有那么的绝对。而且，你们用的是定量的测定么？
【在 b****s 的大作中提到】
: 应该在differentiation时才表达的2个gene，最近一直在proliferation的时候就表达
: 了，重试
: 了很多遍都是这样。
: 开始以为是confluency太高，有部分differentiation，就连续几天控制在5%以下，再
: 取cfl 20%
: 时的RNA，还是发现gene表达了。
: 确定Growth Medium没问题，那2个gene也不是什么新发现，cell line也是实验室经常
: 用的，一筹
: 莫展。。。。。。
|
n********k
发帖数: 2818 | 6 who said this?----coask:)))...nonetheless, it is the mindset of many
including many PIs and reviewers...I could
RT-PCR out many so-called differentiated/non expressed genes in my cells...I
don't think it is a big deal but
some think it is wrong/not pretty:))...but the reality is PCR is sensitive,
if the primers/PCR are very effective,
you probably can get a band with anything:)). theoretically a single copy of
mRNA shall be able to produce a
band, that could be even produced by a chromatin breat
【在 a***e 的大作中提到】
: 应该在differentiation时才表达的2个gene???????????????????????
: who said this?
|
b****s
发帖数: 148 | 7 近十几年的paper都验证过的，我自己都验证过
【在 a***e 的大作中提到】
: 应该在differentiation时才表达的2个gene???????????????????????
: who said this?
|
b****s
发帖数: 148 | 8 这两个是很well-known的gene，一直是用来做control的，忽然最近就表达异常了
用的是PCR，primer和条件都是几年来沿用的
【在 s******y 的大作中提到】
: 一般基因的表达没有那么的绝对。而且，你们用的是定量的测定么？
|
s******y
发帖数: 28562 | 9 Did your lab change serum lately?
In many occasions, the growth factor could lead to abnormal expression of
genes |
b****s
发帖数: 148 | 10 the growth medium is same..
recipe都一样..
莫非serum或L-Glu浓度过高也会让细胞differentiation么?
of
【在 s******y 的大作中提到】
: Did your lab change serum lately?
: In many occasions, the growth factor could lead to abnormal expression of
: genes
|
|
|
h****n
发帖数: 2552 | 11 你用的什么血清，有些细胞只有在特定的血清下才能保持不分化或分化状态。 |
b****s
发帖数: 148 | 12 FBS 和 Cals Serum
【在 h****n 的大作中提到】
: 你用的什么血清，有些细胞只有在特定的血清下才能保持不分化或分化状态。
|
s******y
发帖数: 28562 | 13
Even serum from the same company, if from different batch, could be
different.
If you really want to get to the bottom of this problem, dig out some old
medium or ask the company for an old lot of the serum to test it again.
Yes, definitely. The growth factors in serum will have differentiation
effect on some cell lines.
【在 b****s 的大作中提到】
: the growth medium is same..
: recipe都一样..
: 莫非serum或L-Glu浓度过高也会让细胞differentiation么?
:
: of
|
b****s
发帖数: 148 | 14 我以为只有serum和nutrient不足的时候才导致differentiation..
已经木有旧medium了。。。都是新订的serum新配的，recipe没变，batch肯定变了
如果真的进入differentiation，细胞在数量上应该不会增值才对吧。
可是数量每天都在doubling...
old
【在 s******y 的大作中提到】
:
: Even serum from the same company, if from different batch, could be
: different.
: If you really want to get to the bottom of this problem, dig out some old
: medium or ask the company for an old lot of the serum to test it again.
: Yes, definitely. The growth factors in serum will have differentiation
: effect on some cell lines.
|
s******y
发帖数: 28562 | 15 Your new serum is definitely suspicious then!
Call the company and ask them to send you an old batch, or ask around for
other labs to see if they have the old batch.
Or, order serum from a different company
By the way, don't take things too granted.
"真的进入differentiation，细胞在数量上应该不会增值才对吧" is not
theoretically or practically correct
For example, if your cells differentiated from stem cell into fibroblast, or
from stem cell into some kind of progenitor cells,
the differentiated cells surely will rep
【在 b****s 的大作中提到】
: 我以为只有serum和nutrient不足的时候才导致differentiation..
: 已经木有旧medium了。。。都是新订的serum新配的，recipe没变，batch肯定变了
: 如果真的进入differentiation，细胞在数量上应该不会增值才对吧。
: 可是数量每天都在doubling...
:
: old
|
b****s
发帖数: 148 | 16 Thanks..i'll check the serum.
but it's not stem cells..i still feel the fact of doubling is conflict to
differentiation..
for
fibroblast, or
【在 s******y 的大作中提到】
: Your new serum is definitely suspicious then!
: Call the company and ask them to send you an old batch, or ask around for
: other labs to see if they have the old batch.
: Or, order serum from a different company
: By the way, don't take things too granted.
: "真的进入differentiation，细胞在数量上应该不会增值才对吧" is not
: theoretically or practically correct
: For example, if your cells differentiated from stem cell into fibroblast, or
: from stem cell into some kind of progenitor cells,
: the differentiated cells surely will rep
|
j****a
发帖数: 108 | 17 是不是primer的问题？有些primer如果放置过久，也会有影响。可以拿出stock primer
重新稀释。细胞形态有改变吗？ |
b****s
发帖数: 148 | 18 细胞长得也跟proliferate态一样，没有明显differentiation的样子
试过新稀释的primer，木有效果...
primer
【在 j****a 的大作中提到】
: 是不是primer的问题？有些primer如果放置过久，也会有影响。可以拿出stock primer
: 重新稀释。细胞形态有改变吗？
|
j****a
发帖数: 108 | 19 你测这两个基因表达量时加一个分化细胞样本做对比，它们的表达量有区别吗？ |
