RNA Interference

One of the most revolutionary discoveries in molecular biology in the past decade may well be the discovery of RNA interference (RNAi); a mechanism in the cell biology of many eukaryotes in which fragments of double-stranded ribonucleic acid (dsRNA) interferes with the expression of a particular gene whose sequence is complementary to the dsRNA. RNAi has been applied as an experimental technique to study the function of genes both in vivo and in vitro. Researchers are now trying to use this technology for therapeutic purposes in human disease.

RNAi is an RNA-dependent gene silencing process that is mediated by the RNA-induced silencing complex (RISC). The process is initiated by the RNase-III-like enzyme Dicer, which binds and cleaves long dsRNA to produce short double-stranded fragments with two nucleotide 3’ overhangs on either end, called small interfering RNAs (siRNAs). siRNAs are coupled to RISC complex then binds to a target mRNA in a sequence specific manner by base-paring and induce mRNA target cleavage. The cleaved RNA can be recognized by the cell as being aberrant and then destroyed. Therefore without mRNA there is not any translation to get proteins afterwards. This technique is also referred sometimes as gene “knockdown” as RNAi may not totally destroys the expression of the gene but just somehow turns the genes off , unlike the classic processes of gene “knockout” in which expression of a gene is completely eliminated by removing its DNA sequence.

The great power of RNAi in silencing the gene expressions with high specificity has made this technology an interesting subject for many recent studies, trying to use RNAi as a treatment for HIV, viral hepatitis, cancer and genetic diseases. However the therapeutic applications of this method has not yet been approved and still there is considerable difficulties, including difficulties in efficient delivery and uncertainty about potential toxicity of this method , which are left to be overcome in order to apply in vivo. Also potential side effect of using cellular machinery for directing sequence silencing is the big question that is not answered yet.

The following animation shows the RNAi process. It is from the Nature Reviews RNAi collection:

Western blot

Western blot (also called immunoblot) is a technique to detect specifically one protein in a mixture of large number of proteins and to obtain information about the size and relative amounts of the protein present in different samples.

In this method first proteins are separated using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE).

Then they are moved onto a nitrocellulose membrane. The proteins retain the same pattern of separation they had on the gel.

The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. This step is required as both antibodies and the target are proteins and proteins like to bind to nitrocellulose. By placing the nitrocellulose membrane in a dilute solution of protein, we can prevent the unwanted interactions between the membrane and the antibody used for detection of the target protein as the protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached.

An antibody is then added to the solution which is able to bind to its specific protein and forms an antibody-protein complex with the protein of interest. (In fact there is no room on the membrane for the antibody to attach other than on the binding sites of the specific target protein).

Finally the nitrocellulose membrane is incubated with a secondary antibody, which is an antibody-enzyme conjugate that is directed against the primary antibody.

The location of the antibody is revealed by incubating it with a substrate that the attached enzyme converts to a product that can be seen and followed and then photographed.




Western blot is a confirmatory test for a positive HIV ELISA as it allows one to visualize antibodies directed against different viral protein in a human serum sample.

La Rétrotranscription de l'ARN des Rétrovirus

La Rétrotranscription (the reverse transcription) est une étape importante dans le cycle infectieux des rétrovirus. Elle consiste en synthèse d'un ADN proviral à partir du complexe d'initiation formé d'une amorce ARNt hybridée à l'ARN génomiques des rétrovirus. La rétrotranscriptase, l’enzyme impliqué dans ce processus, est une des principales cibles des médicaments antiviraux.

Dans le film j'ai essayé de montrer les différentes étapes de la rétrotranscription:

ELISA

Enzyme-Linked ImmunoSorbent Assay (ELISA Assay) is a biochemistry technique which is used to determine if a particular protein is present in a protein sample and to quantify how much of the protein is present. ELISA is also used in immunology to detect the presence antibodies or antigens in a given sample.

The ELISA assay utilizes two antibodies: one antibody is specific to the antigen of interest (i.e your protein of interest); and the 2nd antibody is coupled to an enzyme. The second antibody gives the Elisa assay its "Enzyme-linked" name or the EL of ELISA. This secondary antibody catalyzes chromogenic or fluorogenic changes in the presence of a chromgenic or fluorogenic substrate respectively.

Generally there are 5 types of ELISA:

1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
5. Multiplex ELISA

ELISA is used to identify the presence of an antigen, such as a disease-related agent, from a sample of body fluid. This is usually the first test used to detect infection with HIV. If antibodies to HIV are present (positive), the test is usually repeated to confirm the diagnosis. If ELISA is negative, other tests are not usually needed.