Difference between revisions of "20.109(S18):Investigate RNA-seq data using public databases (Day5)"

Introduction

Protocols

Part 1: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on preparing and delivering your Journal Club presentation.

Part 2: Perform qPCR analysis

In a previous laboratory session you purified RNA from your DLD-1 and BRCA2- cells and used the RNA to generate cDNA. Today you will use quantitative PCR to probe the transcription levels of your gene of interest.

Part 2a: Prepare primers

While you were away the sequences for the gRNA you designed were submitted to Integrated DNA Technologies (IDT). IDT synthesized the DNA oligos, or primers, then lyophilized (dried) them to a powder. Follow the steps below to resuspend your oligos.

1. Centrifuge the tubes containing your lyophilized qPCR oligos for 1 min.
2. Calculate the amount of nuclease-free water needed to give a stock concentration of 100 μM.
3. Resuspend each primer stock in the appropriate volume of sterile water, vortex, and centrifuge.
4. Calculate the volume of each stock that is required to prepare a 100 μL of solution that contains each qPCR oligo at a concentration of 10 μM.
   • Try the calculation on your own first. If you get stuck, ask the teaching faculty for help.
5. Prepare a primer mix using the volumes calculated in Step #4.
   • Be sure to change tips between primers!
6. Return the rest of your qPCR oligo stocks and your primer specification sheets to the front bench.

Part 2b: Clean cDNA

1. Retrieve your cDNA samples from the front laboratory bench.
2. Add 100 μL of Buffer PB to each cDNA preparation.
3. Place four QIAquick columns into collection tubes and carefully label each according to your cDNA samples.
4. Transfer the entire volume of cDNA + Buffer PB (~120 μL) from each tube into the appropriate QIAquick column.
5. Centrifuge at maximum speed for 30 s, then discard the flow-through.
6. Add 750 μL of Buffer PE to the QIAquick columns, centrifuge at maximum speed for 30 s, then discard the flow-through.
7. Centrifuge the QIAquick columns at maximum speed for 1 min.
   • This will remove any residual liquid from the columns.
8. Move each QIAquick column into a clean, labeled 1.5 mL centrifuge tube.
   • Be sure to remove the caps so they do not break off in the centrifuge.
9. To elute the DNA, add 50 μL of dH₂O pH = 8 to the center of the column and leave at your benchtop for 1 min.
10. Centrifuge at maximum speed for 1 min.
    • The liquid in the microcentrifuge tube is your cleaned cDNA.
11. Alert the teaching faculty when you have finished and you will be escorted to the NanoDrop to measure the concentration of your cDNA samples.

Part 2c: Prepare cDNA for quantitative PCR assay

In addition to probing your gene of interest and p21 transcript levels, you will also use primers specific for GAPDH. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is an enzyme that catalyzes a step of glycolysis, which serves to break down glucose for energy and carbon molecules. Because GAPDH expression should not be altered by etoposide treatment we will use its transcript levels to normalize the p21 expression levels between the DLD-1 and BRCA2- cell lines. To alleviate some of the workload, the teaching faculty will setup reactions for untreated DLD-1 and BRCA2-/- using the GAPDH, p21, and your primer pairs.

1. Label 12 eppendorf tubes according to the designations below:
   • A = DLD-1 +etoposide, GAPDH primers
   • B = DLD-1 +etoposide +drug, GAPDH primers
   • C = BRCA2- +etoposide, GAPDH primers
   • D = BRCA2- +etoposide +drug, GAPDH primers
   • E = DLD-1 +etoposide, your primers
   • F = DLD-1 +etoposide +drug, your primers
   • G = BRCA2- +etoposde, your primers
   • H = BRCA2- +etoposide +drug, your primers
   • I = DLD-1 +etoposdie, p21 primers
   • J = DLD-1 +etoposide +drug, p21 primers
   • K = BRCA2- +etoposide, p21 primers
   • L = BRCA2- +etoposide +drug, p21 primers
2. Using the concentration measurements from Part 2b, calculate the volume of each sample that contains 20 ng of cDNA.
   • If the volume is greater than 8 μL alert the teaching faculty.
3. Prepare master mixes of your samples such that:
   • Each reaction should contain 20 ng of cDNA, 2 μL of the appropriate diluted primer solution, and water for a total volume of 10 μL.
   • The volume of water is calculated as 8 μL minus the volume calculated in Step #2 for each sample.
   • For each sample/primer set combination, prepare enough master mix for 3.5 reactions.
4. Give the tubes with your master mixes to the teaching faculty

Each master mix will be used to prepare three reactions (10 μL each), such that your samples are run in triplicate. This will allow you to complete statistics when you analyze the data on M2D8. To each 10 μL of sample the teaching faculty will add 10 μL of 2X SYBR Green reagent.

For your reference, the PCR cycling conditions are listed below:

Part 3: Analyze RNA-seq data using public databases

attach exercise...

Reagents

• QIAquick PCR purification kit (Qiagen)
• qPCR primers:
  • GAPDH forward, 5' - GAA AGC CTG CCG GTG ACT AA - 3'
  • GAPDH reverse, 5' - GCC CAA TAC GAC CAA ATC AGA G - 3'
  • p21 forward, 5' - CCA GCT GAG GTG TGA GCA G - 3'
  • p21 reverse, 5' - GTT CTG ACA TGG CGC CTC C - 3'
• 2X SYBR Green (BioRad)

Navigation links

Next day: Journal club I presentations