RNAi and microRNA Method Forum

Cos7 Cells to produce retrovirus

Thu, 19 Nov 2009 11:58:37 -0800

I'd like to find an alternative to Hek293T cells for retrovirus production. I understand that Hek293Ts contain the SV40 and large T antigen. Cos7 also contain these. Would this be a suitable cell type to make retrovirus? I appreciate your insight.

miRNA gene UTRs?

Wed, 18 Nov 2009 15:38:30 -0800

Are there any Drosophila miRNA genes for which 5' and 3' UTRs are known? Or any ESTs of pri-miRNAs that might indicate putative UTRs? We are looking to potentially add endogenous UTRs to our miRNAs to help them stay in the nucleus longer so that they will be more efficiently processed by Drosha, but most websites (e.g. miRbase, flybase) only have transcription info on the actual mature miRNA. Thanks.

Verified RNAi Sequences

Wed, 18 Nov 2009 05:18:13 -0800

Hi everybody,

Does anyone know a database, where I can search for verified RNAi Sequences for siRNA, shRNA and miRNA?

Thanks for any help.

Greetings
AAtRS

Transfection Efficacy

Tue, 17 Nov 2009 15:59:42 -0800

Hello all. I would like to perform transfection optimization using oligofectamine. Invitrogen only have established protocol against HeLa and HUVEC cell line. When designing the method for this optimization, I discovered that most of the kit / paper used fluorophore labelled non targetting siRNA to perform the optimization.

I wonder if I can use the positive control (validated Lamin A/C siRNA) to carry out transfection optimization?Then assuming the Lamin knockdown as my transfection efficacy, I would produce the transfection efficacy data using reverse-transcription pcr (or WB) ?

Thank you for any suggestion!

Question about miRNA-star and Taqman Q-PCR miRNA array validation

Tue, 17 Nov 2009 09:54:30 -0800

Hi. I have 2 questions on miRNAs I was hoping someone here might be able to answer. Firstly do miR-star strands target differential mRNAs to the non-star strand. If so, are these targets accounted for in prediction databases e.g. TargetScan pulls miR family targets.

Secondly, I have conducted a few miRNA Q-PCR arrays which I am currently validating. The problem I have is the RT primers for the array are cyclic (40 cycles) but single assays consist of using a single non-cyclic RT reaction. This causes my endogenous control (u6 or RNU48) to come up much later in the cycles compared to the arrays. So my question is, if my endogenous is CT 26 for U6 in the single assays, can anyone here comment on what CT would be moderately expressed miRNA? I ask this because miRNAs I initially selected from my arrays that were coming up at CT 30 (U6= CT19) are coming up too late for single assay validation (CT 37).

Thanks in advance,

Regards,

Richard

siRNA and plasmid cotransfection

Mon, 16 Nov 2009 14:42:09 -0800

Hi everyone,

I am currently establishing a co-transfection of miRNA inhibitor and plasmid DNA into a colon cancer cell line.
The only thing with which I am currently struggling with is how to set up a proper
pre-test series to figure out the best transfection conditions.
Ultimately I would like to perform my transfection in 6-well plates as I would need
a significant amount of cells to perform additional assay (like migration assay etc.)

Nevertheless I do not want to waste unnecessary material for setting up the assay so
I thought of performing the setup in 96-well plates as this saves me money, reagent and time.

So here is the question that still bothers me about this 96-well setup thing.
How do I scale up the reagent volume from 96 to 6 well then, what parameter to consider?
Just multiply every amount of reagent by 16 or consider the surface area?
I am still wondering if this is the way to go as I think that 96-well plates and
6 well plates are considerably different.
But on the other hand if I have to consider doing replicates for each condition,
I would be using tons of reagent just for setup if I would go with 6 wells for setup...

maybe someone who already did these kind of setup experiments could share how
she/he succeeded.

Thanks in advance!

rna from plasmid

Mon, 16 Nov 2009 01:19:01 -0800

hello everyone,

i am still new in this field and i would like to ask from which link or reference materials can i know the procedure and concept of synthesizing biotinylated rna from plasmid with the gene of interest into insert to the plasmid.

thank you so much.

shRNA stable line

Thu, 05 Nov 2009 05:50:53 -0800

Hello all,
I recently made some shRNA stable lines in U2-OS cells. While the shRNA worked well in transient transfections (>60% reduction with ~60-70% transfection efficiency), the final clones I obtained after two rounds of selection exhibited <25% reduction compared to controls. The shRNA plasmid also has pCMV driven GFP expression, so I can see that all of my clones (~30) have clonal GFP expression, so integration occurred. I am assuming that the copy number is too low compared to the transient transfection, since GFP fluorescence is so sensitive. Any ideas on how to improve the copy number? Should I use higher concentrations of antibiotic for selection?

T cell transduction

Tue, 03 Nov 2009 20:39:01 -0800

I want to transduce human Tcells with a Foxp3 shRNA. I've heard that Tcells are very hard to transduce, does anyone have any tips?

retrovirus shRNA

Sat, 31 Oct 2009 02:37:15 -0700

hi,
Can someone suggest a good retroviral plasmid for expressing shRNA?

preferably the selection marker can be easily cut out and replaced with fluoroscent proteins.

can it be packaged in ecopacks/amphopacks?

thanks.

design parameters for multiple siRNA expression in vector

Thu, 29 Oct 2009 00:59:34 -0700

Hi everyone..
I wish to express siRNAs through vector.
my questions are
Is it possible to add sequences of more than one siRNA in tandem in a vector??
If yes then what should be the desired distance between the two selected siRNA to be diced properly by dicer??
Is their any specific feature of substrate (i.e.dsRNA) required for dicer to recognize n make a staggered cut to give mature siRNA or it acts on dsRNA without any particular recognition pattern..??

Why U6 promoter is always used in siRNA vectors... ??

ur replies will be of kind help as i am new in this field...

regards
nipun

microRNA AAAS webinar

Wed, 28 Oct 2009 13:57:42 -0700

miRNA research design webinar hosted by AAAS. Event Date: November 10, 2009 12 noon Eastern, 9 a.m. Pacific, 5 p.m. GMT

Register here: http://www.sciencemag.org/webinar/

Pronounciation of miRNA

Wed, 28 Oct 2009 06:53:54 -0700

Hi,

I'm new to miRNA field.
First i would like to know the correct way to pronounce miR-17-2?
It is microRNA-seventeen-two or microRNA-one seven-two?

Thanks

miRNA TARGETS

Tue, 27 Oct 2009 08:15:18 -0700

Hi all,
I am writing a proposal for my preliminary exam and I have a question. I am very new to this field of micro-RNA.
I wanted to know if I tested that my micro-RNA targets a gene in HeLa cells by luciferase assay, will the micro-RNA surely target this gene in my cell line of interest.
In case of HeLa cells, I will be transfecting the synthetic micro-RNA too. However, in my cell line of interest HBMVEC, I know this micro-RNA is expressed. So should I also do a luciferase report analysis in HBMVEC along with Hela cells?
I appreciate your help and time.
Thanks a lot.

3'UTR constructs

Mon, 26 Oct 2009 14:59:08 -0700

Hi All,

We are a little tight on time right now, trying to roll a study. I have a few miRs of interest all predicted to target the same gene. Cloning the 3'UTR and then performing the studies might take a while. A quick google search gives 2 companies- GeneCopoeia and SwitchGear, that presumably sell pre-made 3'UTR constructs tagged to Luciferase reporter for my gene of interest.

I haven't noticed any publications citing their constructs. Just wondering if anyone had any experience with either of these 2 companies. Or if you knew any other that worked well for you.

Also, a related question, most Luc-reported studies use a microRNA expression plasmid co-transfected with their Luc-reporter plasmid. I was wondering if there is any reason that co-transfection with miR-mimics (eg: Pre-miRs from Ambion) are not popular for repression of Luciferase signal?

Any help is appreciated. Thanks!

Concatemerization of miRNA...

Fri, 23 Oct 2009 13:51:10 -0700

I get 2 bands on agarose gel on running my qPCR products for some miRNAs whereas a few others show just one band around 25bp(expected size). I believe the ones for 2 bands are mature and precursor miRNAs. Has anyone seen concatemerization of some miRNAs whereas some others not showing this??

Thanks!!

is dsRNA stable in cells?

Wed, 21 Oct 2009 09:18:26 -0700

Hi,

I'm doing some RNAi experimets with the malaria mosquite, and I have some doubts.

I'm using dsRNA targeted for a couple of genes, my question: is this dsRNA stable for a couple of days, or is it all degraded to siRNA by DICER, I'm asking this because I have some primers that anneal in the same region of the gene and I afraid of getting wrong expression levels if I use these in qRT-PCR to asses the efects of silencing. because I might be also getting amplification of the dsRNA injected

thanks in advance

Co-transfection of miRNA and vector expressing target sequence

Tue, 20 Oct 2009 14:00:33 -0700

Hi,

I am new to this forum and the research field. I would appreciate if someone could help me with this query. I want to do a co-transfection of miRNA with a vector expressing a target sequence? Could someone help with the ratio of miRNA:vector DNA or suggest where I could find it? I am using Metafectene Pro as the transfection reagent in a 6-well plate.

Thank you!

How to overexpress star miRNA (miRNA*) from pre-miRNA construct

Sun, 18 Oct 2009 19:25:09 -0700

How to overexpress star miRNA (miRNA*) from pre-miRNA construct

I am working on miRNA* field. I plan to clone the pre-miRNA (about 500 bps with flank sequence) into vectors, then express mature miRNA in which the miRNA* is only by product. But, how to construct the pre miRNA vector in which the MAIN PRODUCT is miRNA*, not mature miRNA. I have no any clue on it and no papers on it.

Thanks in advance

Who are the big names in miRNA research?

Sat, 17 Oct 2009 08:37:57 -0700

Hello everyone

Bit of a strange request this one, my boss wants me to compose a list of the big names in miRNA research at the moment.
I'm writing a miRNA based grant and she would like me to find a big name collaborator to boost our chances of funding as we don't have experience with miRNAs in our lab.

Any suggestions very welcome.
BW
Emily

P.S. We are based in the UK