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)?
-
-
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. -
Hi Luc,
Thanks for your reply! The re-amplification is only for one or two samples (out of hundred; and are from different biological replicates) which failed to get enough yield.Comment
-
CRISPR/Cas9 sparked the gene editing revolution for both research and therapeutics.1 But this system still showed severe issues that limited its applications. The most prominent were the heavy reliance on PAM sequences, delivery limitations, double-stranded breaks that prompt unintended edits and cell death, and editing inefficiency (both in targeting and in knock-in reliability).
Despite this, “CRISPR helped turn genome editing from a specialized technique into...07-31-2026, 11:01 AM -
Proteomics platforms are evolving rapidly, with advances in mass spectrometry and affinity-based approaches expanding what researchers can detect and at what scale. As the field moves toward deeper proteome coverage and clinical applications, scientists face an increasingly complex landscape of tools. This article will explore how researchers are navigating these choices to find the right platform for their work.
The systematic characterization of the human proteome has...07-20-2026, 11:48 AM
Comment