How can I shear RNA from formaldehyde-fixed cell lysates with Covaris M220?

I'm having some trouble obtaining an RNA 200-500 base fragment size in lysates from cells fixed with 1% formaldehyde. I've based the parameteres of my protocol on the Covaris protocol for Total...

10 November 2015 8,328 1 View

What is the stability of PLGA in chloroform?

I was hoping to prepare a long term stock of PLGA in chloroform, which I can store at -20C. Would anybody know what the long term stability of PLGA in chloroform would be? Thanks

06 July 2015 6,842 4 View

Secondary size peak for PLGA nanoparticles. Why?

I'm producing ~200 nm siRNA PLGA/DOTAP nanoparticles using the double-emulsion solvent evaporation technique. When I sized the particles using DLS the majority (95%) were of the correct size...

05 June 2015 7,195 9 View

How can we incorporate uncertainty in repeat experiments?

The authors of the following paper make some excellent points about the use of and difference between repeats and replicates: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321166/ They are very...

03 April 2014 2,346 13 View

Anyone have experience with trizol (phenol:chloroform) extraction while excluding small RNAs?

I'm aiming to attempt the opposite of what many on this forum are trying. I'm seeking to perform Trizol extraction with the implicit aim to purify RNA species that are longer than small RNA...

01 February 2014 9,203 3 View

Are there any reagents that can produce inter-RNA crosslinks that are reversible without having to introduce special nucleotides to the RNA?

Seeking information on Reversible RNA-RNA crosslinking

06 July 2013 7,100 2 View

Binding strength antibody vs oligonucleotide?

Would anybody perhaps know how an antibody compares to a short (~25 nt) oligonucleotide in terms of binding strength. I'm aware that this depends on many variables, but I'm just trying to get a...

06 July 2013 6,974 8 View

Biotinylated oligonucleotide probe degradation?

I'm performing a RNA-precipitation protocol with specific biotin-TEG labelled probes which bind streptavidin magnetic beads. Over time my enrichments have become significantly lower. My suspicion...

05 June 2013 9,181 1 View

Aptamers or oligonucleotides?

A number of groups use aptamer-conjugated RNA sequences to specifically isolate RNA binding proteins. What is the advantage of using aptamers over e.g. using oligonucleotides to pull-down those...

04 May 2013 6,143 5 View

qPCR: Standard deviation of reference sample?

I've come across various methods to determine the summarised error in qPCR experiments that included all biological and technical replicates. Although I haven't had much trouble performing these...

04 May 2013 4,889 27 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

Ester cleavage conditions?

Hi all, I need to cleave the ester in Ethyl 2-amino-1H-imidazole-5-carboxylate to make the carboxylic acid and retrieve the product from solution to use in downstream applications of linking to...

01 March 2021 8,766 3 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

Unknown debris sheets observed in cell culture flasks?

Hi, We culture neural progenitor cells in T175 culture flasks coated with PLO-Laminin. We have successfully grown these cells with our protocol for months. Recently, we observed large sheets...

25 February 2021 3,443 4 View

What is a good reagent for lentiviral plasmid transfection in HeLa cells?

I have been using Lipofectamine 2000 but was hoping there may be a better protocol or one for something like Lipofectamine PLUS reagent.

24 February 2021 1,633 4 View

How to prepare a thin polymer film by post-polymerization/crosslinking?

24 February 2021 2,362 5 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