How do you interpret this? What does it mean for wet-lab side of things, does it inform any step that went right or wrong? This is for a total RNAseq minus rRNA experiment.
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Hi seqtechno1,
Issues could be:
-Ribodepletion Kit used does not target tRNAs. If they are targeted then they will not be removed. Most kits use a pretty high (2.0x or above) bead cleanup, so even very small tRNAs would make it through the ribodepletion process. You could reduce the bead ratio so that tRNAs are removed but mRNA stays (assuming your mRNA is high enough quality to not be mixed in with the tRNAs.
-If this is the case, and most of your rRNA deleted RNA is tRNA, then when you go to do cDNA synthesis, most of your cDNA will be from tRNA, not your desired mRNA. Most RNASeq library prep kits will have various bead cleanups at various ratios. If you want, you try reducing the bead cleanup ratios at these steps as well to try and remove as much small cDNA products synthesized from small tRNA as possible.
These ideas will only work if your mRNA is go high enough quality to not be mixed in with tRNAs. IE if you are working with FFPE Total RNA, then its a lot harder to troubleshoot.
Latest Articles
Collapse
-
by seqadmin
The field of immunogenetics explores how genetic variations influence immune responses and susceptibility to disease. In a recent SEQanswers webinar, Oscar Rodriguez, Ph.D., Postdoctoral Researcher at the University of Louisville, and Ruben Martínez Barricarte, Ph.D., Assistant Professor of Medicine at Vanderbilt University, shared recent advancements in immunogenetics. This article discusses their research on genetic variation in antibody loci, antibody production processes,...-
Channel: Articles
11-06-2024, 07:24 PM -
-
by seqadmin
Next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) are essential techniques for investigating the genome, transcriptome, and epigenome. In many cases, choosing the appropriate technique is straightforward, but in others, it can be more challenging to determine the most effective option. A simple distinction is that smaller, more focused projects are typically better suited for qPCR, while larger, more complex datasets benefit from NGS. However,...-
Channel: Articles
10-18-2024, 07:11 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 11-08-2024, 11:09 AM
|
0 responses
217 views
0 likes
|
Last Post
by seqadmin
11-08-2024, 11:09 AM
|
||
Started by seqadmin, 11-08-2024, 06:13 AM
|
0 responses
160 views
0 likes
|
Last Post
by seqadmin
11-08-2024, 06:13 AM
|
||
Started by seqadmin, 11-01-2024, 06:09 AM
|
0 responses
80 views
0 likes
|
Last Post
by seqadmin
11-01-2024, 06:09 AM
|
||
New Model Aims to Explain Polygenic Diseases by Connecting Genomic Mutations and Regulatory Networks
by seqadmin
Started by seqadmin, 10-30-2024, 05:31 AM
|
0 responses
27 views
0 likes
|
Last Post
by seqadmin
10-30-2024, 05:31 AM
|
Comment