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
Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...-
Channel: Articles
03-22-2024, 06:39 AM -
-
by seqadmin
The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.
Avian Conservation
Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...-
Channel: Articles
03-08-2024, 10:41 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 03-27-2024, 06:37 PM
|
0 responses
13 views
0 likes
|
Last Post
by seqadmin
03-27-2024, 06:37 PM
|
||
Started by seqadmin, 03-27-2024, 06:07 PM
|
0 responses
11 views
0 likes
|
Last Post
by seqadmin
03-27-2024, 06:07 PM
|
||
Started by seqadmin, 03-22-2024, 10:03 AM
|
0 responses
53 views
0 likes
|
Last Post
by seqadmin
03-22-2024, 10:03 AM
|
||
Started by seqadmin, 03-21-2024, 07:32 AM
|
0 responses
69 views
0 likes
|
Last Post
by seqadmin
03-21-2024, 07:32 AM
|
Comment