We are preparing RNA-Seq libraries for Illumina HiSeq 3000. However, a few of the libraries are low in concentration (<5 nM, tested on Agilent Bioanalyzer High Sensitivity DNA chip) and we can not go back for RNA isolation. Can we take aliquot of 2 ul from the library and re-amplify for 5 cycles (and bead purify) to ensure proper yield? And can we use this data for following analysis (compare gene expression levels)?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Hi Jin1,
I assume you will be pooling multiple libraries? ; thus, the molarity of the final pool will have to meet the requirements of your sequencing service -- not the individual libraries.
If you also have higher concentrated libraries it is very likely that the final concentration will be no problem.
I would very much avoid amplifying a subset of the libraries in additional PCR reactions and cleanups - this likely could introduce technical variation. I would rather amplify all if absolutely needed.
Latest Articles
Collapse
-
by seqadmin
The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...-
Channel: Articles
05-06-2024, 07:48 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, Yesterday, 02:06 PM
|
0 responses
7 views
0 likes
|
Last Post
by seqadmin
Yesterday, 02:06 PM
|
||
Started by seqadmin, 05-14-2024, 07:03 AM
|
0 responses
27 views
0 likes
|
Last Post
by seqadmin
05-14-2024, 07:03 AM
|
||
Started by seqadmin, 05-10-2024, 06:35 AM
|
0 responses
47 views
0 likes
|
Last Post
by seqadmin
05-10-2024, 06:35 AM
|
||
Started by seqadmin, 05-09-2024, 02:46 PM
|
0 responses
59 views
0 likes
|
Last Post
by seqadmin
05-09-2024, 02:46 PM
|
Comment