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Forum: Bioinformatics 04-12-2018, 01:42 PM
Replies: 0
Views: 604
Posted By renuka22
TMM normalisation and p-value

Hi all,

I have all the TMM normalised data from 3 replicates of RNA-seq done by someone. But I am not sure about the data analysis carried out based on these counts. For example - Some genes have...
Forum: Bioinformatics 11-15-2017, 12:34 PM
Replies: 1
Views: 1,096
Posted By renuka22
RNA seq bioinformatics

Hello all,

I have RNA seq data from 2 replicates of control and treated (say for example C1,C2,T1,T2). But when I compared the replicates separately (C1vsT1) ,(C2vsT2) there were around 1500...
Forum: Sample Prep / Library Generation 06-09-2017, 09:58 AM
Replies: 1
Views: 954
Posted By renuka22
rna purification

Hello,

I have RNA concentration around 15-18 ng/ul (260/280 is 1.5-1.8 but 260/230 is very low ~0.5) on which I need to do polyA library preparation and sequencing. The company said you need to...
Forum: General 05-19-2017, 09:41 PM
Replies: 5
Views: 2,417
Posted By renuka22
Does doing polyA treatment lowers RIN values? My...

Does doing polyA treatment lowers RIN values? My polysomal RNA values showed good RIN values of 9.5..but when I did the polyA, it was in the range of 5-6. The concentration could not be detected by...
Forum: General 05-19-2017, 09:34 PM
Replies: 5
Views: 2,417
Posted By renuka22
No, I have never used this. Would have been a...

No, I have never used this. Would have been a good idea though..
Forum: Sample Prep / Library Generation 05-12-2017, 09:19 AM
Replies: 15
Views: 3,188
Posted By renuka22
Is it a good idea to pool 2 rat litters together...

Is it a good idea to pool 2 rat litters together to get more RNA and solve the yield issue? Will it affect the RNA sequencing data?
Forum: Sample Prep / Library Generation 04-18-2017, 08:38 PM
Replies: 15
Views: 3,188
Posted By renuka22
I will be doing high sensitivity assay on qubit....

I will be doing high sensitivity assay on qubit. No idea if it will detect anything. But a company has agreed to do rna seq if the samples are >20pg. They mentioned they have a kit which can be...
Forum: Sample Prep / Library Generation 04-18-2017, 09:21 AM
Replies: 15
Views: 3,188
Posted By renuka22
jteeee2 - Yes I am planning to do qPCR on the...

jteeee2 - Yes I am planning to do qPCR on the samples to confirm. No, I have not done Dnase treatment, I did not want to go through extra treatments due to yield issues. Thanks for the suggestions. I...
Forum: Sample Prep / Library Generation 04-17-2017, 08:28 PM
Replies: 15
Views: 3,188
Posted By renuka22
jteeee2 - Thanks for your suggestion..I have some...

jteeee2 - Thanks for your suggestion..I have some samples for which the bioanalyzer is not able to detect any rna., might be there is no rna or very less. Please find attached the results from the...
Forum: Sample Prep / Library Generation 04-17-2017, 01:13 PM
Replies: 15
Views: 3,188
Posted By renuka22
Hello, Perhaps people don't know much about...

Hello,

Perhaps people don't know much about polysomal RNA...

pmiguel - Yes you are right. Polysomal RNA is rich in rRNA. We have not used sucrose gradient due to yield issues and after doing...
Forum: Sample Prep / Library Generation 04-12-2017, 11:27 AM
Replies: 12
Views: 2,678
Posted By renuka22
Is it a good idea to do DNAse treatment because I...

Is it a good idea to do DNAse treatment because I got to know that the yield I will be getting from this tissue after clean up will be around 20ng/ul. I am planning to do a polyA isolation later and...
Forum: Sample Prep / Library Generation 04-12-2017, 09:06 AM
Replies: 12
Views: 2,678
Posted By renuka22
Ok. Thanks. Do you have any idea why are the Zymo...

Ok. Thanks. Do you have any idea why are the Zymo columns so cheaper than Qiagen. Which one is good? Zymo also has some clean up kits with DNAse in those. Do those work good?
Forum: Sample Prep / Library Generation 04-12-2017, 08:05 AM
Replies: 12
Views: 2,678
Posted By renuka22
Ok got you. I think it would be better to do a...

Ok got you. I think it would be better to do a clean up first and then measure concentration. Can the DNAse also be a source of degradation because I have not done the DNase treatment? I don't have...
Forum: Sample Prep / Library Generation 04-11-2017, 01:10 PM
Replies: 12
Views: 2,678
Posted By renuka22
Here is the nanodrop image..

Here is the nanodrop image..
Forum: Sample Prep / Library Generation 04-11-2017, 01:05 PM
Replies: 12
Views: 2,678
Posted By renuka22
Hi, I think you are absolutely right. There...

Hi,

I think you are absolutely right. There is a peak at 270nm seen on nanodrop. Is there any way I can clean up the samples with a kit. Will that help? What can be a good solution to this...
Forum: Sample Prep / Library Generation 04-11-2017, 09:13 AM
Replies: 12
Views: 2,678
Posted By renuka22
Hi, Sorry about the missing information. I...

Hi,

Sorry about the missing information. I checked on nanodrop and it showed concentration in the range of 200-300 ng/ul. I am working on rat primary retinal ganglion cells and isolating polysomal...
Forum: Sample Prep / Library Generation 04-10-2017, 12:15 PM
Replies: 12
Views: 2,678
Posted By renuka22
RNA degradation?

Hello,

PFA the electropherogram image of RNA degradation on bioanalyzer. There were no peaks from samples which I can conclude as no RNA or RNA has degraded completely. But the RIN value is 3.4....
Forum: Bioinformatics 04-04-2017, 11:22 AM
Replies: 4
Views: 2,238
Posted By renuka22
So I guess the p-value is between technical...

So I guess the p-value is between technical replicates hence meaningless. Thanks a lot!
Forum: Sample Prep / Library Generation 04-04-2017, 11:10 AM
Replies: 15
Views: 3,188
Posted By renuka22
Poly A and RNA seq

Hello,

I have some polysomal RNA samples, and I need to send them for polyA isolation and RNA seq (polyA selection). Does anyone know any good companies that can do polyA isolation and further...
Forum: Bioinformatics 04-03-2017, 07:35 AM
Replies: 4
Views: 2,238
Posted By renuka22
Ok. But can the p-value be between my control and...

Ok. But can the p-value be between my control and treatment samples as this is what they mentioned in their analysis and p<0.05 means those genes are differentially expressed?

Thanks
Forum: Bioinformatics 04-02-2017, 05:32 PM
Replies: 4
Views: 2,238
Posted By renuka22
p-value and fold change

Hello,

I got my rna-seq analysis from the company and they have assigned p-value to each gene. Is it possible to have a p-value from only one set of data since I had sent only one batch and there...
Forum: General 03-30-2017, 08:10 AM
Replies: 5
Views: 2,417
Posted By renuka22
polyA library preparation and RIN values

Hi there,

I am new to this forum and had some questions about the RNA bioanayzer data and nanodrop readings. I got a good concentration of 200-300ng/ul when I analysed my samples by nanodrop...
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