O*********e
发帖数: 79 | 1 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! |
S*****s
发帖数: 287 | 2 How did you precipitate RNA? If you use ethanol wash, did you get rid of
ethanol completely? I have tried both LiCl and Ethanol precipitation, then
resolve in either pure water or TE, no problem at all.
I
Thanks
【在 O*********e 的大作中提到】
: 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!
|
O*********e
发帖数: 79 | 3 It was ethanol precipitation, 100% first and then wash with 70%
I've been dealing with RNA for quite some time, never had this problem
before.
The RNA was from peritoneal macrophages. I was warned before about the tough
RNA pellets, and just realized it was such a pain ...
【在 S*****s 的大作中提到】
: How did you precipitate RNA? If you use ethanol wash, did you get rid of
: ethanol completely? I have tried both LiCl and Ethanol precipitation, then
: resolve in either pure water or TE, no problem at all.
:
: I
: Thanks
|
w******e
发帖数: 1187 | 4 how did you isolate the RNA? trizol? did you use enough? is the pellet
oily?
I had similar problem before.
I
Thanks
【在 O*********e 的大作中提到】
: 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!
|
a****k
发帖数: 1130 | 5 don't let the RNA pellet dry completely, it'll greatly decrease its
solubility
I
Thanks
【在 O*********e 的大作中提到】
: 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!
|
O*********e
发帖数: 79 | 6 trizol
i think I used plenty
I can see the pellets floating in the TE, not oily though
I did the isolation with trizol samples from other cells, they were just
fine (nice pellets, dissolved easily in TE), so I'm thinking it's the
macrophage RNA that gives me trouble.
anybody on this board working with macrophages?
【在 w******e 的大作中提到】
: how did you isolate the RNA? trizol? did you use enough? is the pellet
: oily?
: I had similar problem before.
:
: I
: Thanks
|
w***a
发帖数: 4361 | 7 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.
【在 O*********e 的大作中提到】
: trizol
: i think I used plenty
: I can see the pellets floating in the TE, not oily though
: I did the isolation with trizol samples from other cells, they were just
: fine (nice pellets, dissolved easily in TE), so I'm thinking it's the
: macrophage RNA that gives me trouble.
: anybody on this board working with macrophages?
|
n***w
发帖数: 2405 | |
a*****i
发帖数: 1045 | 9 I only use water to dissolve.....then it disappears |
O*********e
发帖数: 79 | 10 天，这个都会上首页。。
多谢wdgca的回复，我也觉得是macrophage的问题。下次用那个kit试试看
不是干透的问题，我提RNA已经有4年了，从来不用speed vacuum，都是风干的，pellet
一变透明就加TE。TE和water我都用过，做microarray的样品用nuclease-free water，
其他没那么高质量要求的时候就用TE，没发现什么区别。还有我前面说过，我同时提的
macrophage和splenocyte的RNA，后者一点问题都没有，macrophage的RNA pellet其实
还小些，但就是不溶。昨天折腾了好久，还是能看到薄薄的一片pellet飘在管子里，但
测 OD到了100ng/ul，足够一个反转录PCR了,就将就着用了。下次一定试试那个Qiagen
RNeasy Kit。也给以后要提macrophage RNA的同学提个醒，really tough pellet，要
是样品宝贵的话不要自己提，直接上kit。 |
Y**I
发帖数: 444 | 11 vortex到手酸。
俺曾经vortex RNA pellet40分钟。。。最终被击破。
pellet
Qiagen
【在 O*********e 的大作中提到】
: 天，这个都会上首页。。
: 多谢wdgca的回复，我也觉得是macrophage的问题。下次用那个kit试试看
: 不是干透的问题，我提RNA已经有4年了，从来不用speed vacuum，都是风干的，pellet
: 一变透明就加TE。TE和water我都用过，做microarray的样品用nuclease-free water，
: 其他没那么高质量要求的时候就用TE，没发现什么区别。还有我前面说过，我同时提的
: macrophage和splenocyte的RNA，后者一点问题都没有，macrophage的RNA pellet其实
: 还小些，但就是不溶。昨天折腾了好久，还是能看到薄薄的一片pellet飘在管子里，但
: 测 OD到了100ng/ul，足够一个反转录PCR了,就将就着用了。下次一定试试那个Qiagen
: RNeasy Kit。也给以后要提macrophage RNA的同学提个醒，really tough pellet，要
: 是样品宝贵的话不要自己提，直接上kit。
|
O*********e
发帖数: 79 | 12 pfpf~ 哈哈
【在 Y**I 的大作中提到】
: vortex到手酸。
: 俺曾经vortex RNA pellet40分钟。。。最终被击破。
:
: pellet
: Qiagen
|
