Shreffler:RTPCR

Overview

This protocol is for RT-PCR of JAX project samples. The protocol has been optimized for these samples, but may be generalized as noted in several sections. The volume/tube in the table is calculated against 3.0 - 4.0µl of RNA. For example, for 7.5µl of RNA, multiply all variables by ≈2.

Materials

• 10X RT Buffer
• 25 mM MgCl₂
• dNTPs Mixture (2.5 mM)
• oligo dT primer
• RNase Inhibitor (20 U/μL)
• MultiScribe Reverse Transcriptase (50 U/μL)
• Tissue Culture Grade DMSO

Procedure

Component	Volume/Tube (μL)	Final Concentration
RNase-free water	Variable to make total = 20 μL	--
10X RT Buffer	1.0	1X
25 mM MgCl2	2.2	5.5 mM
dNTPs Mixture (2.5 mM)	2.0	500 μM/dNTP
oligo dT primer	0.5	2.5 μM
RNase Inhibitor (20 U/μL)	0.2	1.25 U/L
MultiScribe Reverse Transcriptase (50 U/μL)	0.25	1.25 U/μL
Total	≈6.15/tube	--

Note: If changing the reaction volume, make sure the final proportions are consistent with the recommended values above.

‡ Add Rnase free water to the total volume above to bring the total volume to 10µl.

§ Random Hexamers, oligo d(T)16, or sequence-specific reverse primers can be used for primers of cDNA synthesis.

Note: The RT reaction volume can vary from 10µL to 100µL. Increasing the RT reaction volume will reduce the total number of reactions.

1. Make a mixture of H₂O and RT buffer in the same eppendorf tube according to the table above.
2. Add MgCl₂ into the mixture in the eppendorf tube
3. Add dNTP to the mixture in the eppendorf tube.

Note: Use the Oligo-dT and not the Hexamer.

1. Add RNase Inhibitor to the mixture.
2. Add the Reverse Transcriptase Enzyme (RT) into the mixture. Place mixture on ice.
3. Use 3.0 - 4.0μL of RNA for the RT experiment as indicated above.
4. Use a pipette or a repipetter to dispense between 6 - 7μL of mixture (cocktail) to the eppendorf tube containing the RNA to make a final volume of 10µl.

Note: For better mixture, fast spin in a Microcentrifuge for 5-10s

1. Transfer the tubes to a Thermocycler. Set the Thermocycler using the RT pre-programmed, or set the Thermocycler according to the instructions in the table below:

Step	Incubation	RT	Inactivation
Time	10 min	30 min	5 min
Temperature	25 °C	48 °C	95 °C

Note: It can be stored overnight at 4ºC.

Make PCR cocktail as follows:

'	100X	550X
PCR Buffer	100	550
MgCl2	50	275
dNTPs	10	110
Primers	10+10	->
SYBR	5	55
Taq	5	55
dH2OPrimers	270	1485

Note: The volume prescribed for each reagent for the PCR cocktail is intended for 36 tubes or samples.

1. Add dH₂O (1.48 mL) to a 50 mL tube on ice.
2. Add 550 μL of PCR buffer (from Taq Polymerase, PCR buffer, and MgCl₂ kit). Store MgCl₂ at 4°C.
3. Add 275 μL of MgCl₂ into tube on ice.
4. Add (110 μL of dNTP mix). The dNTP is at a [100 μM]. Resuspend to [5µM]. Note: All four dNTPs are added into one (1) eppendorf tube.
5. Make SYBR Green by diluting from 1000X into 100X in DMSO and H₂O. 990 μL of sterile DMSO + 10 μL of SYBR Green. The working 200X diluted stock is prepared by making a 1:1 dilution of Cybergreen with dH₂O (mix 500µl of Cybergreen into 500µl of H2O (1:1 dilution)).
6. Add 55 μL of 200X diluted SYBR Green to the PCR buffer, H₂O, and MgCl₂ cocktail on ice. Gently vortex. Keep on ice.
7. Add 55 μL of Taq polymerase to the cocktail. Gently vortex, and keep on ice.
8. Thaw frozen Primers kept in the -80ºC freezer, or make Primers. Primers arrive from IDT as lyophilized contents of a tube, they are then resuspended in sterile dH₂O to a concentration of 0.1 nM/μL (this is the stock solution). The working solution is made by mixing 10 μL of primer with 90 μL dH₂O. The final concentration of the Primers should be at 5µM.
9. Take 10 μL of the forward (5' - 3') and reverse primers (3' - 5') of each gene of interest and add into the tubes containing the enzyme mix.
10. Add 230 μL of PCR cocktail to each tube containing both the forward and the reverse primers.
11. Align your PCR strip tubes.
12. Add 60 μL of dH₂O to 10 μL of the cDNA (RT reaction).

Note: Optimal cDNA dilution should be @ 1:6 fold dilution, thus if cDNA reactions were in 10 μL, add 60 μL dH₂O.

1. Add 80μL dH₂O to the no template control strip tube.
2. Spin each PCR tube down for 5 – 10s in a microcentrifuge.
3. After spin, transfer the PCR mixture in each tube into the appropriate PCR strip tube, and cap.
4. Place PCR tube in a rack. Keep on ice until analysis.

Discussion

discuss this protocol

References

1. pmid=

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare