It's not really accurate to say that one RNAi technology is better than others. They all have advantages and disadvantages, and which you choose should depend on your question. What are you trying to knock down, and where in the cell do you expect the target RNA to be located? Are you working in cultured cells or in vivo? How long do you need the target to be suppressed for? I have no experience with the specific siRNA you're looking at, but for a first attempt at an RNAi experiment, going with a product that has a company backing it up (and established protocols) is probably a good plan.

For controls, the absolute minimum is to use a non-targeting control which lacks complementarity to any RNA in your species of interest. For high confidence in your results, compare a no-transfection (or saline injection) control, a non-targeting control, and two different siRNAs targeting the same RNA.

For validation, confirm by qPCR that expression of your target is greatly reduced after 48-72 hours. Depending on the target, acceptable knockdown might be anything over 50%, with 75% and better usually considered good. If your target encodes protein, confirm by Western blot that expression of the protein is also reduced. Depending on the half-life of the protein, you might need to wait a few more days to see good knockdown at the protein level.

Good luck! :)

I would propose to take a look on published protocols, e.g. via PubMed (as adequate source).

Hi Sandeep,

we have been developing a set of negtaive control siRNA (AllStars Negative Control siRNA) and positive controls that should fit pretty well. The AllStars Negative control has been designed to have minimal non specific effects (we checked on Affy GenChip arrays) and tested to be functional as shown in reporter assays using the suitable target motif.

The positive control siRNAs should make optimization of transfection parameters easy. To do so, we generated siRNA pools targeting ubiquitously expressed genes that are essential for cell survival. After successful transfection with this control, a high degree of cell death is induced within 48 - 96h after transfection which can be detected by simple light microscopy. Chosing conditions inducing high degree of cell death with AllStars Cell death control but no cytotoxicity by AllStars negative control helps quickly finding best transfection parameters.

Hope that helps!

Best

Peter

Dear Sandeep,

For publication purposes, a journal will ask you to show knock down data from 2 different targeting siRNAs against your gene of interest. Some journals will ask you to show the knock down in 2 different cell lines.

For the negative controls you will need a non-targeting siRNA such as a scrambled siRNA sequence of the target sequence which should not target any gene in your host cell line or you can use siRNA against exogenous genes such as an siRNA agains GFP, Luciferase, beta-galactosidase, etc....

All of the above should be verified at the mRNA level by qPCR or at the protein level by western blotting provided that the protein is not stable during the time frame of the experiment as mentioned by Laura Leighton above.

Thank you Peter Hahn and Abdullah A Shaito for your suggestions.

Thank you Laura Leighton .

I am using Human Monocyte cell line (THP 1)and targeting a gene expressed on Phagosomal/endosomal membrane.

I want to infect my cells with Mycobacterium tuberculosis. So, at what stage I should transfect ( I am thinking about infection after transfection). Please suggest.

Are two siRNA and a negative control enough or do i need more siRNA and positive control as well.

I will take established items from Thermo fisher and will do qPCR and western later to validate.

thank you.

infection should be done after transfection. For better results it may be a good idea to establish a stable THP-1 cell line using shRNA of your gene of interest.

If you know a very good positive control then use it, otherwise 2 siRNA and a negative control are enough.

Sandeep R. Kaushik I'm not familiar with your cell type or with the time course of infection with M. tuberculosis. You should determine how long a single transfection with the siRNA you're looking at will suppress a gene for, and decide whether that is enough time to infect your cells and have them reach the endpoint you need. If it's not, Abdullah A Shaito has a good suggestion about establishing a cell line which stably expresses shRNA to suppress the gene over the full time course of your experiment.

There's not really a positive control for most siRNA experiments, the closest you can get to that is validating multiple siRNAs targeting the same gene. Another good orthogonal experiment is to knock down a gene which you know or believe is downstream of your gene of interest and show that the phenotype is similar.

Silencer Select siRNA works great. use positive and negative control, and deliver with Lipofectamine 2000 or 3000

You could also try our advanced siRNA reagent: https://www.sitoolsbiotech.com/sipools.php

Hi Sandeep!

Probably you already have your answer! But I am looking at siRNA transfections protocols, and what I noticed is usually big companies have many protocols with different types of cells and lineages.

Further, I found a beneficial paper that explains the general transfections technologies and methods!

I hope it worked!

The paper:

Article Transfection types, methods and strategies: A technical review

Cell Lignages protocol (Thermo)

https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html