Happy new year, everyone. I am posting to receive any advice you have for me regarding an issue I'm having with RiboZero plant leaf! I am using the kit for RNA extracted from Oryza sativa. I've attached the Nano trace for DNase-treated samples and the Pico trace for RiboZero treated samples. It looks like there is basically nothing left after RiboZero, at least for the case of the first 4 samples. The last two look like incomplete subtraction, or, something else went wrong. Any tips?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
This is really a question for Epicentre tech support. But, for what it is worth:
(1) We generally get extremely low yields of non-rRNA from plant total RNA compared to animal rRNA.
(2) 90% of the time when there is some protocol of this sort -- dealing with dilute solutions of RNA or DNA that produces a poor yield -- it is the result of attempting to recover that RNA or DNA via alcohol precipitation. Sadly it seems few people are taught that this this methodology is difficult and has a high failure rate in all but the hands of the most talented bench scientist. So if you see a protocol calling for you to precipitate RNA or DNA solutions at concentrations likely to be below 50 ng/ul, look for an easier method. Spend an extra few dollars on a zymo column rather than wasting weeks (or longer) trying to get the alcohol precipitation to work.
--
Phillip
-
Originally posted by pmiguel View PostThis is really a question for Epicentre tech support. But, for what it is worth:
(1) We generally get extremely low yields of non-rRNA from plant total RNA compared to animal rRNA.
(2) 90% of the time when there is some protocol of this sort -- dealing with dilute solutions of RNA or DNA that produces a poor yield -- it is the result of attempting to recover that RNA or DNA via alcohol precipitation. Sadly it seems few people are taught that this this methodology is difficult and has a high failure rate in all but the hands of the most talented bench scientist. So if you see a protocol calling for you to precipitate RNA or DNA solutions at concentrations likely to be below 50 ng/ul, look for an easier method. Spend an extra few dollars on a zymo column rather than wasting weeks (or longer) trying to get the alcohol precipitation to work.
--
Phillip- Gina
Comment
-
Hi Gina,
Maybe too late but this week I made a trial library starting the Ribozero with ~10ng! It worked except for some primer dimers, the library was there (Scriptseq V2, Epicentre) as seen by Bioanalyaer and PCR on 3 different genes. Look for my recent question (no-one replied to see the attached curves.
Good luck,
Guy
Comment
-
I also succeeded in making libraries out of very little starting material.
I face another problem: The riboZero reaction is inconsistent and sometimes doesn't work at all.
These are my parameters: 2.5 µg of RNA, 8 µl of removal solution. the sample is young leaf of rice and I use the RiboZero plant leaf kit.
Anyone had difficulties?
Comment
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, Yesterday, 06:37 PM
|
0 responses
10 views
0 likes
|
Last Post
by seqadmin
Yesterday, 06:37 PM
|
||
Started by seqadmin, Yesterday, 06:07 PM
|
0 responses
9 views
0 likes
|
Last Post
by seqadmin
Yesterday, 06:07 PM
|
||
Started by seqadmin, 03-22-2024, 10:03 AM
|
0 responses
49 views
0 likes
|
Last Post
by seqadmin
03-22-2024, 10:03 AM
|
||
Started by seqadmin, 03-21-2024, 07:32 AM
|
0 responses
67 views
0 likes
|
Last Post
by seqadmin
03-21-2024, 07:32 AM
|
Comment