Question

Procedure

I. Reverse Transcription. For each of your RNA samples set up the following reaction: *Remember to use proper RNA handling technique! 1. In a 0.2 ml size RNAse-Free tube add the following:

• 0.5 µg RNA (no more than 6 µl) You can add as little as 0.1 µg of RNA if your concentration is low • 2 µl dT primer • Nuclease Free Water to final volume of 8 µl * Before you start write the exact volume that you will add of each reagent in your notebook. 2. Mix and briefly centrifuge 3. Incubate at 70°C for 5 min *This step denatures secondary structures that might be present in your RNA. Because RNA is single-stranded, bases on the same strand can form bonds folding the RNA and inhibition the reverse transcription reaction 4. Immediately after removing tubes from the 70°C incubation, centrifuge briefly and place tubes on ice. *If samples are allowed to cool slowly, the RNA may refold to form secondary structures. 5. Add the following to each tube from step 1 (keep your tubes on ice!): • 10 µl of 2X Reaction Mix* • 2 µl M-MuLV Enzyme Mix • Mix and briefly centrifuge * The reaction mix contains dNTPs, Buffer and RNAse inhibitor 6. Mix and briefly centrifuge Biol 313L Labs 2-4, Part II 3 7. Incubate the reaction at 42°C for one hour 8. Inactivate the enzyme by incubating at 80°C for 5 min 9. Centrifuge briefly and place tubes on ice. 10. Add 30 µl sterile water. ⇒ You have now made cDNA from your RNA. The cDNA is not prone to degradation by RNAses so you no longer have to use RNA technique after this point in the procedure. II. PCR reaction Refer to Part I of the lab manual for a general description of the PCR procedure. For each of your cDNA samples, you will prepare 3 PCR reactions to assay for expression of 3 genes in each tissue. This makes a total of 6 PCR reactions. 1. Prepare 6 0.2 ml tubes. Number your tubes 1-6. Important! Also label your strip of tubes with your initials or some other identifiable way. 2. Make a table in your notebook that denotes which primer set and which cDNA you will add to each tube. Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Primers cDNA 3. To each PCR tube add: • 12.5 µl GoTaq 2X Master Mix* • 2 µl gene-specific Forward primer, 2.5 µM Mix** • 2 µl gene-specific Reverse primer, 2.5 µM Mix** • 3 µl cDNA • 5.5 µl Sterile water * The GoTaq Master Mix includes: Buffer, dNTPs (ATP, TTP, CTP, GTP) and Taq DNA polymerase. ** Be sure to only add the primer mix for the gene you plan to amplify. 4. Keep your tubes on ice after you complete setting up the reaction. These will be put in the thermocycler (PCR machine) when enough groups have their reactions set up Biol 313L Labs 2-4, Part II 4 5. The PCR cycles will be programmed in the Theromocycler. Your TA will start the reactions for you. The following cycles will be run: • 95°C, 3 min • 95°C, 30sec • 59°C, 30sec • 72°C, 1 min 6. The PCR reactions will run for between 1 and 2 hours. You your TA will remove the PCR reactions from the machine when it is complete and store the samples in the freezer until next lab.

1.) At the end of the reverse transcription reaction (Part I) what is in your tube?

2. Explain why each primer is used: What is the oligo-dT primer for in the RT reaction and what is the purpose of the primer pairs used in the PCR reactions? 30 cycles Biol 313L Labs 2-4, Part II 5

3. At what step are you producing DNA that corresponds to a specific gene? At what step are you producing DNA that corresponds to all of the mRNAs (expressed genes) within the tissue?

Answer 1

1.

Reverse transcription produces cDNA from the mRNA sample.

So, cDNA and residual reaction components (mRNAs, dNTPs, buffer components and enzyme) are left in the tube at the end of the RT reaction.

2. Eukaryotic mRNA contains poly-A tail. SO, the poly-T primer binds to the complementary poly-A tail and extends the primer to synthesize cDNA from the mRNA template.

Poly T primer is non-specific. It binds to all mRNAs that contain poly-A tail.

Primers that are designed to bind specific genes can not bind all genes. During PCR reaction, they bind to their target gene and amplify the flanking sequence.

3.

During reverse transcription step, cDNA that corresponds to all of the expressed genes (mRNAs) within the tissue are produced.

During PCR step with a specific set of primers, a particular target gene is amplified.