RT-PCR Protocols

cDNA Synthesis
Real-time PCR Reaction Setup
Real-time PCR Run
One-Step RT-PCR
Preparation of DNA for Standard Curve
Primer Sequences for Internal Control Genes
Sequencing RT-PCR Products

cDNA Synthesis 

1.  Bring up 5 µg of total RNA in 7 µl H2O ¹
2. Add 5 µl primer (100 ng/µl) ²
3. Denature at ~85° for 3 minutes. Put on ice then spin briefly
4. Add 4 µl First Strand 5x Buffer, 2 µl 0.1 M DTT and 1 µl 15 mM dNTP ³
5. Mix thoroughly and incubate at 42° for 2 minutes to bring samples to correct temperature
6. Add 1 µl Superscript II reverse transcriptase ⁴ (200 U/µl). Incubate at 42° for ~1 hour.
7. Inactivate at ~85° for 10 minutes
8. Dilute cDNA dependent upon how much RNA you have available. We have used dilutions up to 1:17 with success 

Notes
This protocol is essentially the method provided by Invitrogen for use with Superscript II reverse transcriptase. 1µl RNasin (20,000-40,000 U/ml) may be added to the reaction mix to ensure mRNA integrity.
Once the cDNAs are made they have to be validated. We recommend that each cDNA be first tested using primers for the cyclophilin A gene. For each cDNA that is to be used in a comparison, the level of cyclophilin A gene expression should be essentially constant. 

¹ Use PCR quality H2O
² Typically, use random hexamers (Rn6) (Invitrogen #48090-011)
³ Amersham Pharmacia Biotech #27-2035-01
⁴ Invitrogen Superscript enzyme #18064-022, which comes with 5x buffer and 0.1 M DTT

Real-Time PCR Reaction Set-Up

1. Design primers to produce amplicons in the range 150 to 300 base pairs ¹
2. Set up the following reaction mixture at room temperature in the following order
   1 µl        primer.up (6.25 µM)
   1 µl        primer.dn (6.25 µM)
   10 µl        Qiagen Quantitect Sybr Green-Enzyme and Dye mixture
   8 µl        1:16 cDNA
   20 µl        final volume per well 

Prepare a master mix for each set of cDNAs. Typically each sample point will be performed in triplicate so it is usually easiest to create master mixes that are then aliquoted into a 96-well plate. Always prepare enough master mix for one extra reaction because of pipetting errors. For example, for a triplicate mix you will prepare a master mix for 4 reactions:
          4 µl        primer.up (6.25 µM)
          4 µl        primer.dn (6.25 µM)
          40 µl        Qiagen Quantitect Sybr Green-Enzyme and Dye mixture
          32 µl        1:16 cDNA
 
Notes
The only kit we use is the Quantitect SYBR Green PCR Kit (Qiagen #204143). 96-well plates (MJ Research plates: #MLL-9651 White; caps: #TCS-0803 Clear) work significantly better than strips, apparently giving a more even transfer of heat to each sample.

₁ Use the Primer 3 program (http://primer3.ut.ee/). In our hands we have an ~90% success rate with primers designed using this program

Real-time PCR Run Parameters

Something similar to the following program should be used for the MJ Research Opticon:
 
95°C        15 min
94°C        30 sec
55°C        30 sec
72°C        1 min
??°C**        10 sec   (**This is the plate read temperature.)
Plate Read
Go to line 2 for 39 times more
Perform melting curve from 65 to 95; read every 0.2°C; hold for 5 s between reads;
Incubate at 12°C forever
End
 
The plate read temperature is determined empirically. For a typical experiment, you must first determine whether the primers work and the actual melting temperature of the amplicon. In this trial experiment the plate read temperature is set at a low level, typically 68°C. In the actual experiment, the plate read temperature is set just below the melting temperature of the amplicon.

One-step RT-PCR Protocol
 
This protocol has been designed for use of the Qiagen QuantiTect SyBr Green RT-PCR Kit in a DNA Engine Opticon machine. The reagent volumes are given for a 20 µl reaction. If desired, these volumes can be scaled up for a 50 µl reaction.

1. Make sure the appropriate masterfile is loaded on the cycler, so that the machine is ready to go right after the plate is loaded.
2. Thaw all reagents and keep on ice, except for the Quantitect RT Mix that has to be taken from -20°C immediately before use, always kept on ice and returned to -20°C immediately after use.
3. Prepare a mastermix for the desired number of samples + 1 as follows:
   Volumes/sample:
   1 µl        primer.up (25pmol/µl)
   1 µl        primer.dn (25pmol/µl)
   10 µl        2x QuantiTect SyBr Green Mix
4. Pipette 18.5ul of the mastermix in each tube/well.
5. Add the following to each tube/well
   0.2 µl        QuantiTect Reverse Transcriptase Mix (add first)
   1 µl        RNA (add last)
6. Spin the plate or tube strip down briefly and put back on ice
7. Load samples immediately in the Cycler and start the following program
   MJ Research Cycler Program:
   1.    50°C        30 min
   2.    95°C        15 min
   3.    94°C        30 sec
   4.    55°C        30 sec
   5.    72°C        30 sec
   6.    ??°C ¹        10 sec
   7.    Plate read
   8.    Go to line 2 for 39 times more
   9.    Perform melting curve from 65 to 95; read every 0.2°C; hold for 5 s between reads;
   10.    Incubate at 12°C forever
   11.    End 

For troubleshooting, please refer to the Qiagen QuantiTect SyBr Green RT-PCR Handbook available online.

¹ Insert appropriate Plate read temperature

Preparation of DNA for Standard Curve

To quantitate the results it is necessary to create a standard curve. The nature of the plasmid used for the standard curve is not important but the melting temperature of the standard amplicon should be relatively high so that the read temperature can be set optimally for the amplicon produced in the experimental samples. It is best to avoid using a plasmid that contains a gene that is of interest in the experiment because this can produce contamination problems.

1. Dilute stock plasmid 1:1000 to a dilution of 1ng/µl.
2. Prepare standards:
   1 ng -    dilute 1 ng/µl stock solution 1:8 :
             70 µl stock DNA solution + 490 µl PCR-grade water
   0.1 ng -    dilute 1 ng/8 µl standard solution 1:10 :
             50 µl standard + 450 µl PCR-grade water
   0.01 ng -    dilute 0.1 ng/8 µl standard solution 1:10 :
             50 µl standard + 450 µl PCR-grade water
   0.001 ng -    dilute 0.01 ng/8 µl standard solution 1:10 :
             50 µl standard + 450 µl PCR-grade water

Primer Sequences for Internal Control Genes

The following primers have been successfully designed and used by the UDSF staff in the indicated species. Primers have only been tested in brain and heart tissues and might not be adequate for use on samples from other tissues/cell lines, although from the same species. 

* Important: the values for plate read temperatures given above are valid only when using the Qiagen QuantiTect SyBr Green PCR and RT-PCR Kits only. If you are using other commercial or handmade kits, please verify these temperatures experimentally before running an experiment.

¹ "Upstream" or "forward" primer
² "Downstream" or "reverse" primer

Sequencing RT-PCR products

1. Dilute the given rtPCR product 1:100 in a stock with PCR water
2. Use a 25 µM dilution of primer. This higher concentration of primer works better for the sequencing reaction
3. Combine reagents in the following order in 0.2ml tubes:
   8 µl of PCR water
   3 µl of primer (25 µM)
   1 µl of rt-PCR product (1:100 dilution) 

Notes
Always run sequencing samples in duplicates, some sequences will fail. The sequences will look noisy because of the residual primers in the rtPCR product.

Enter the names of the samples in the DNA Sequencing Facility customer computer in the order of one strip tube at a time