SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Maximum number of cycles for kit Miseq 50 cycles MisUser Illumina/Solexa 8 10-18-2017 10:28 PM
Smart-Seq v4 ultra low RNA kit seqgirl123 Sample Prep / Library Generation 4 03-16-2017 02:39 PM
2x150 cycles vs 2x250 cycles: coverage, quality, aligning and cost issues seqask Illumina/Solexa 3 08-31-2013 07:27 AM
Has any one here used 200 cycles SBS kit to perform reads shorter than 50 cycles? Lanlan Illumina/Solexa 2 02-27-2012 08:54 PM

Reply
 
Thread Tools
Old 05-07-2017, 11:56 PM   #1
candida utilis
Junior Member
 
Location: Taiwan

Join Date: May 2017
Posts: 5
Default SMART Seq v2: RNA amount and cycles for preampfification

Hi all,
We aim to compare differential mRNA expression between 2 different cell types. We sorted 2 populations of cells by FACS, extract RNA by column based methods, and SMARTSEQ v2 for pre-amplificaiton of cDNA.

Between those cell types, A has very low cell number (150-500 cells). In contrast, B has higher cell number (50,000 cells) but its RNA amount is less. So, I only do RNA quantification for B, because RNA amount in A is too low to be detected by Ribogreen.

For Pre amplification of cDNA, I have some questions:

(1) If the starting RNA amount used for SMARTSeq V2 is different between samples, will it affect the RNA Seq results, if we aim to compare the differential mRNA expression between those samples?

(2) For the pre-amplification of cDNA, will the number of PCR cycles for A and B need to be the same, if I would like to compare the differential mRNA expression between those cell types? If sample A need 20 cycles to get sufficient cDNA, while sample B only need 15 cycles, what should I do? Thank you
candida utilis is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 11:43 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO