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All seats are full and registrations have been closed.
Regulation of Gene Expression: quantitative
Regulation of Gene Expression: quantitative
Short Description: Quantifying the levels of bacterial gene expression using Real time qPCR (SYBR-based)
Type: Hands-on
Duration: One day (9.00 AM to 5.00 PM)
Fees: INR 2596 (INR 2200 + 18% GST)
Category (for payment): MBY Workshop Tier 2
Aimed at: College students (B.Sc., M.Sc.) and PhD
Application Process: Click here for the details
Registration form (This link will be different for each workshop)
Application process (in brief)
Application process (in brief)
Check the above table to figure out whether suitable dates are available or not.
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We verify your payment and send Registration Confirmation Email. Your registration is now complete.
We strongly encourage the applicants to look at the detailed application process before paying the fees and filling the form.
Course Content
Course Content
1. Basic laboratory practices and RNA handling practices
1. Basic laboratory practices and RNA handling practices
This will include basic safety precautions, handling of micropipettes and safe disposal of laboratory wastes along with precautions taken while working with RNA samples.
To know more about how to handle micropipettes, watch this video or practice using this simulation. For best RNA handling practices before and after extraction, please read the article here.
2. Synthesis of cDNA by reverse transcription
2. Synthesis of cDNA by reverse transcription
Total RNA of known concentration is used (given) to synthesize complementary DNA (cDNA) using MMLV reverse transcriptase enzyme. The first strand of cDNA is then used as a template for gene specific RTqPCR. To know more about cDNA synthesis, watch this video.
3. In silico primer designing and MIQE guidelines
3. In silico primer designing and MIQE guidelines
Understand what are MIQE guidelines and how one goes about primer designing.
4. Testing RTqPCR primer efficiency (e value) using a standard curve
4. Testing RTqPCR primer efficiency (e value) using a standard curve
The standard curve is generated by setting up reactions using different dilutions of the same DNA template. This is followed by data interpretation and efficiency calculation.
5. Quantification of relative gene expression levels (SYBR-green based)
5. Quantification of relative gene expression levels (SYBR-green based)
Once the entire RNA has been converted cDNA, expression of the gene of interest needs to be looked at. One of the ways to do this is through Real Time quantitative Polymerase Chain Reaction (RTqPCR). This not only amplifies the number of copies of the gene of interest but also quantifies that number in real time after each PCR cycle.
Relative expression of the gene of interest in treatment group is calculated against a control group using the delta delta Ct (ddCt) method and appropriate normalization gene is used as an internal control. To know more about RTqPCR, you can either read this external link to article or watch this video.
6. Data interpretation and melt curve analysis
6. Data interpretation and melt curve analysis
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