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 field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...-
Channel: Articles
04-22-2024, 07:01 AM -
-
by seqadmin
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...-
Channel: Articles
04-04-2024, 04:25 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, Yesterday, 08:47 AM
|
0 responses
12 views
0 likes
|
Last Post
by seqadmin
Yesterday, 08:47 AM
|
||
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
60 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
60 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
54 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
Comment