How can I tag mRNA by cloning inside a cell?

Hi I am interested to check a particular mRNA and a protein interaction/ localization inside cells. For this reason, I want to tag the transcript. Beside RNA-FISH or any such hybridization...

10 November 2017 8,697 0 View

In there any report on Lipofectamine RNAiMAX mediated transfection of CRISPR-Cas9 plasmid in THP-1 cells?

02 March 2017 4,713 1 View

Any suitable protocol for Ni-NTA based pull down and interaction study?

Hi..I have over-expressed a protein (tagged with His) via retroviral transduction method in THP-1 cells. Now I am interested to check if there is any binding partner with my tagged protein in a...

11 December 2016 2,637 4 View

How can I separate ribosome-free cytosol using sucrose cushion?

I intend to separate ribosomal and non-ribosomal fractions of cytosol using 20% sucrose cushion. I have already run the cell lysate on top of the 20% sucrose solution for 2 hrs at 1,39,000 g. I...

05 June 2016 1,102 2 View

How can I search the expression of particular set of genes in NCBI GEO database?

I am interested in searching the gene expression profile of a particular set of genes in a specific disease condition like atherosclerosis from NCBI GEO database. As I have almost no background in...

06 July 2015 615 7 View

Why CD11b and CD14 mRNA expression is getting down after 6hrs of rest?

I have converted THP1 monocytes to macrophage using 5 ng/ml PMA for 48 hrs in complete RPMI1640.PMA Containing media was discarded and cells were washed with sterile DPBS for 3 times and given...

03 April 2015 5,086 2 View

Why are my PMA differentiated THP1 cells not responding to LPS induction?

I have differentiated THP1 monocytes into macrophage using 5 ng/ml PMA for 48 hrs. Then after washing twice with DPBS i have kept the diffrerentiated cells for 3 hrs in serum free media. Then I...

02 March 2015 1,298 8 View

Why are my THP1 cells forming clumps?

I am culturing THP1 cells in RPMI 1640 (as advised by ATCC formulation, except 2ME). Unfortunately my cells are forming clumps a few hours after seeding (at 1* 10^5 cells/ml) before even going to...

02 March 2015 154 10 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 View

Why is the efficiency of viral mRNA electroporation in PBMC low?

Hello, I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of...

27 February 2021 9,233 3 View

What is the best concentration of cDNA to be used for qPCR?

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul...

24 February 2021 8,618 3 View

Recommendations on homogenizers for RNA isolation?

I will be performing RNA isolation with frozen lung biopsy samples and am in the market for a tissue homogenizer. I am wanting a homogenizer that will fit in a 1.5ml tube. I was looking at the...

24 February 2021 3,623 3 View

Genescan gives 3 peaks instead of one?

Hi all, I am doing a genescan analysis for a deleted exon in the patient sample. The amplified region is 215 bp. In the patient sample because of homozygous deletion, no peak is observed while in...

23 February 2021 5,645 1 View

RNA-seq DEGs downstream analysis and reporting in literature?

I used EdgeR to get the list of DEGs (differentially expressed genes) from my liver RNA-seq experiment. I have done the gene overrepresentation and GSEA based pathway analysis. I am currently...

23 February 2021 8,969 1 View

Is it efficient to do liquid biopsy to investigate the gene expressions of the tumor?

To do liquid biopsy and look for specific gene expressions there are so many published papers. Thera are different protocols and procedures used to isolate circulating cell free RNA or mRNA from...

23 February 2021 7,326 5 View

RNA degraded after good nanodrop readings?

23 February 2021 8,850 2 View