The GE2 Plate with rack and tube of BC4 included in this kit are shipped on dry ice and must be stored at -20 ºC upon receipt. When stored properly at -20 ºC, their quality is guaranteed for 6 months.

The aluminum foil films sealing and the compression mat can be stored at room temperature.

Shelf Life: All reagents are stable for 6 months after receipt if stored at the recommended temperature.

RT² HT First Strand Kit
Catalog no.	C-08
Number of 20 µl reverse-transcription reactions	96
Components	# Included
GE2 Plate with rack: 96-well Plate Pre-loaded with gDNA Elimination Buffer (6 µl per well)	1
12-well Strip Tubes	1
Tube of BC4 (RT Master mix): 650 µl	1
Rack for GE2 Plate	1
Aluminum Foil Sealing Films	2
Compression mat	1
User manual	1

• Please read through this entire protocol before beginning your experiment.
• RNA samples are very sensitive to RNase digestion; therefore, wear gloves and maintain an RNase-free work area while performing this protocol.

Considerations of RNA amount to be used:

The RT² HT First Strand Kit yields results with as little as 25 ng or as much as 5 µg total RNA per well reaction. However, the optimal amount of starting material depends on the relative abundance of the transcripts of interest. Lower abundance transcripts require more RNA; higher abundance transcripts require less RNA. Greater amounts of input total RNA yield a greater number of positive calls; that is, genes expressed in the linear dynamic range of the method. It is also important to use a consistent amount of total RNA for all samples in a single experiment to be characterized and compared.

1. Remove the GE2 Plate from -20 ºC storage and leave at room temperature for 3 minutes.
2. Centrifuge the GE2 Plate at 1000 rpm for 1 min.
3. Put the GE2 plate on the provided rack.
   1. Remove the strip-caps carefully
   2. Add 8 µl RNA sample to each well with a multi-channel pipette and mix by pipetting up and down
   3. Completely seal the plate with the aluminum foil sealing film.
      Note: The amount of total RNA should be in the range of 25 ng to 5 µg.
4. Centrifuge the plate at 1000 rpm for 1 min.
5. Incubate the plate at 37 ºC for 5 min (or room temperature for 10 min).
6. Remove the aluminum seal carefully
7. Aliquot RT Master Mix (BC4) into provided strip tubes
   1. If using a 12-channel micropipettor, aliquot 53 µl per tube
   2. If using 8-channel micropipettor, aliquot 80 µl per tube
8. Pipette 6 µl BC4 (RT Master Mix) from the Strip Tube with a multi-channel pipette to each well of the plate and mix by pipetting up and down
   1. Completely seal the plate with a new piece of aluminum foil sealing film.
9. Centrifuge the plate at 1000 rpm for 1 min.
10. Perform reverse transcription (this can be performed in a regular PCR cycler)
    1. Set up a program for 42 ºC 15 min, 95 ºC 5 min, 4 ºC forever (without multiple cycling)
    2. Put the plate in the PCR cycler with the reusable compression mat on top of the plate
    3. Close lid of the PCR instrument, and run the program.
11. Hold the finished reaction on ice until ready to use for real-time PCR, or place at -20 ºC for long-term storage.

Note: For real-time PCR, the cDNA should not be more than 5% of the total PCR volume.