QRT-PCR

'''Ben Carter, 9/24/2014. Updated 2/23/18.'''

Note: RNAse-free technique must be used for this protocol.

''We use a QIAgility liquid dispensing robot to set up the 96-well plates for qPCR. A multi-channel pipette can be substituted.''

Required Materials

 * RNeasy Plant Mini Kit (Qiagen Cat# 74903)
 * RNA Clean & Concentrator Kit w/DNAse (Zymo) Freeze the DNAse in aliquots.
 * M-MLV Reverse Transcriptase (Life Technologies Cat# 28025-013)
 * Random Hexamers (IDT Cat# 51-01-18-26)
 * Power SYBR Green Master Mix (Life Technologies Cat# 4368706)
 * 50µL Conducting Filter Tips (Qiagen Cat# 990512)
 * MicroAmp Optical Adhesive Film (Applied Biosystems Cat# 4311971)
 * MicroAmp Fast Optical 96-Well Reaction Plate (Applied Biosystems Cat# 4346906) OR MicroAmp Fast Optical 8-Tube Strips (Applied Biosystems Cat# 4358293)

RNA Isolation and Purification

 * 1) Homogenize the tissues using mortar and pestle or bead beater technique. Keep homogenized samples on ice.
 * 2) Extract RNA using the RNeasy Plant Mini Kit according to manufacturer's instructions.
 * 3) Purify the samples using the Zymo RNA Clean & Concentrator Kit with DNAse I treatment according to manufacturer's instructions.
 * 4) Measure the concentrations of the purified RNA samples using a NanoDrop. Samples should have a concentration of 25 to 250ng/µL. Expected 260/280 values are between 2.0 and 2.25. RNA samples should be stored at -20°C or -80°C.

Reverse Transcription using M-MLV Reverse Transcriptase
Note: To obtain 25µM Random Hexamers, suspend 10nmol Random Hexamers in 400µL of RNAse-free water.
 * 1) Add the following to a PCR tube:
 * 2) * 250ng of RNA (maximum volume of 10µL)
 * 3) * 2uL of 25µM Random Hexamers (IDT)
 * 4) * 1uL of 10mM dNTPs
 * 5) * Nuclease-free H2O to 13µL
 * 6) * You can use less than 250ng for very dilute samples, but the cDNA sample is not guaranteed to amplify well in the qPCR reactions.
 * 7) Mix each solution by pipetting with a P20 set to 10µL until there is no turbidity. Incubate at 65°C on a thermal cycler for 5min.
 * 8) Incubate at 4°C on a thermal cycler for 4min.
 * 9) Add 4µL of 5X First-Strand Buffer and 2µL 0.1M DTT. Mix each solution by pipetting with a P20 set to 16µL until there is no turbidity.
 * 10) Add 1µL of M-MLV. Mix each solution quickly but gently by pipetting with a P20 set to 16µL until there is no turbidity. The M-MLV is sensitive to temperature, so keep it on ice.
 * 11) Amplify using the following thermal cycler program:
 * 12) * 25°C for 10min
 * 13) * 37°C for 50min
 * 14) * 70°C for 15min
 * 15) Dilute cDNA samples with 130µL of Milli-Q water. cDNA samples should be stored at -20°C. If you used less than 250ng of RNA template, you must scale down the final solution volume proportionally.

qPCR using the QIAgility Robot and the StepOnePlus Thermal Cycler
''Note: The StepOnePlus assigns arbitrary Ct thresholds with each run. In order to compare results across multiple runs, you must manually normalize the Ct thresholds in the software for all of the runs that you wish to compare.''
 * 1) If the primers are still a lyophilized powder, centrifuge them briefly on a tabletop microcentrifuge. Examine the tube or specification sheet and locate the amount of primer in nanomoles . Multiply this number by 10 to obtain the volume of nuclease-free water to add to the tube. This will yield a 100µM stock solution. Primer stocks should be stored at -20°C or 4°C.
 * 2) For each primer, combine 25µL of the 100µM primer stock from Step 1 with 75µL of nuclease-free water to make a 25µM working primer solution.
 * 3) Prepare a master mix for each gene to be amplified in 1.5mL tubes as follows. (Volumes should be multiplied by the number of cDNA samples you are using. Designed for two replicates per sample. If you have less than 5 samples, make enough for one additional sample to account for the robot's residual volume requirements.)
 * 4) * 6.67µL of Milli-Q water
 * 5) * 0.259µL of the 25µM forward primer working solution
 * 6) * 0.259µL of the 25µM reverse primer working solution
 * 7) * 14.375µL of Power SYBR Green Master Mix
 * 8) Gently pipette the master mixes up and down until there is no turbidity. The listed primer amounts are for 900nM concentrations. Adjust the volumes as appropriate if you desire a different primer concentration. Master mixes and cDNA samples do not need to be kept cold during the robot and qPCR steps.
 * 9) Thaw the desired cDNA samples and vortex them briefly. Tap the tubes on the bench to collect the liquid at the bottom. Remove a MicroAmp Fast Optical 96-Well Reaction Plate or 8-Tube Strip from the package. Select the appropriate QIAgility method file or make your own as per the manufacturer’s instructions. Make sure the tip re-use setting is set to 3.
 * 10) Bring the following materials to the QIAgility robot:
 * 11) * Two 96-well plate holders and your MicroAmp plate or tube strip
 * 12) * cDNA samples and master mixes
 * 13) * MicroAmp Optical Adhesive Film
 * 14) * 50µL Conducting Filter Tips
 * 15) * Flash drive with your QIAgility method file
 * 16) Turn the QIAgility robot’s power to ‘on’ in the back. The robot’s front-panel blue LED will turn on. Open your method file on the connected computer.
 * 17) Place your tips, cDNA samples, plate/tubes, and master mixes in the appropriate locations on the robot. The microfuge tube caps should be open and not occluding any nearby tubes. Ask for help if you have questions about this.
 * 18) Ensure that the tip waste container is empty. Make sure all of the components are calibrated in the software (no red ‘!’ marks). Run the method file.
 * 19) Carefully remove the plate and cover it with the optical film using the brown spreader. OR Remove the optical tube strip(s) and close the caps.
 * 20) Centrifuge the plate/tubes in a 96-well plate centrifuge. Use a second tray and plate/tubes as a balance. Use the short spin option and allow the centrifuge to reach max speed (~4600rcf).
 * 21) Insert the plate into the StepOnePlus instrument. Amplify as per manufacturer instructions.