What are the Problems you face during Next Generation Sequencing Data Processing ? Please Reply atleast one.

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Can MicroRNA cross the blood brain barrier?

In diseased conditions, many genes and gene products are visible in the bloodstream. So my question is whether we can find traces of microRNA which is involved in disease and disorder in blood serum?

24 February 2021 7,064 3 View

Are exogenous molecules transferred between human cells during doubling ?

Hello RG community, I wonderer whether exogenous molecules (e.g. reporter plasmids, miRNAs or other artificial molecules) are transferred between cultured human cells during their doubling ? So,...

21 February 2021 6,633 3 View

Has anyone encountered any problems with abm good microRNA primers for quantification of mature miRNAs?

I've been trying to quantitate mature microRNAs in 293T cells with and without 'X'. 2ug of total RNA (isolated by Trizol method) is converted to cDNA using All-in one microRNA cDNA synthesis kit...

17 February 2021 8,986 2 View

What is the best way to fix plasma derived miRNA for subsequent cDNA synthesis?

I need to quantify blood plasma derived miRNA through q-RT-PCR. For this I need to fix RNA content in my cDNA reaction. What is the best way to do this? Currently, I take readings of my plasma...

03 February 2021 9,330 3 View

What is the general miRNA yield one gets from the Qiagen miRNEASY serum/plasma advanced kit?

I am using the Qiagen miRNEASY serum/plasma advanced kit, using 400ul of frozen plasma. What is the yield you get on average? I am using nanodrop currently for quantification. I am getting...

02 February 2021 8,806 2 View

Which miRNA arm is a right target for analysis, 3p or 5p?

I'm working on selecting mouse miRNAs to analyze and stumbling upon one confusing issue, some of the papers do not specify which arm they analyzed and they just state something like " miR-34a was...

16 January 2021 3,792 4 View

Is negative controls necessary for western blotting?

I am trying to confirm my miRNA target by western blotting. I have a ds mimic and a ss inhibitor of my miRNA. Is it necessary to use negative controls for mimic and inhibitor? If yes, how can I...

09 January 2021 2,320 3 View

Can I predict miRNA by knowing the mRNA target?

Hello! I am trying to predict in silico the miRNA by knowing the target. Most of published research starts by knowing the miRNA and predicts the target using online tools. Are you familiar with...

31 December 2020 7,433 5 View

What are the possible drawbacks of using miRNA sponge to enhance the specificity of gene expression?

I am searching for miRNA repeats put between WPRE and 3'LTR of my lentivirus, in order to enhance the specificity of transgene expression in human neuron cells. But if clinically translation of...

26 December 2020 9,921 2 View

Amit Kumar Singh

Dear Emilian & Gael, All the shared problems can be solved by using physical mapping technology, from OpGEN. OpGEN compliments NGS data & help in refining the data, filling the gaps, align the contigs more accurately etc. It just help you in getting excellent data.

David Kang

From what little I know I believe different types of next gen sequencing result in different problems. Roche 454 Pyrosequencing seems to be bad with indels, but gives you nice long reads. Illumina Solexa seems to be good with indels, but bad with short sequence repeats and have high substitution errors due to modified polymerase and reverse transcriptase. If you look at ABI-SOLiD it is easier to tell between a sequencing error vs a polymorphism, but again you have the problems associated with a short sequence read.