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RT² HT First Strand Kit (96)

Catalog Number Product Description Price  
330411  For 96 samples
Description
The RT² HT First Strand Kit provides a rapid and convenient procedure for efficient first-strand cDNA synthesis and genomic DNA elimination in your RNA samples. It enables simultaneous and easy parallel processing of 96 RNA samples for reverse transcription. The synthesized cDNA is ready-to-use in real-time PCR expression analysis of multiple genes when used with SABiosciences' RT² SYBR Green® qPCR Master Mixes and RT² qPCR Primer Assays, Biology-on-Arrays, or with other experimental procedures requiring cDNA from a large number of samples.
Materials Included / Packing List
Please check the kit components immediately after you receive this package. SABiosciences is not responsible for missing items not reported within two (2) business days upon receipt.

Shelf Life: All reagents are stable for 6 months after receipt if stored at the recommended temperature.
RT² HT First Strand Kit
Catalog no. 330411
Number of 20 µl reverse-transcription reactions 96
 
Components # Included
Tube of GE2 Solution (Genomic DNA Elimination Buffer): 750 µl 1
5 ml Reagent Reservoir 2
Tube of BC4 Solution (RT Master Mix) : 750 µl 1
Blank 96-Well Plate 1
Foil Adhesive Sealing Film 2
Compression Mat 1

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

The foil sealing films, 96-well plate, compression mat, and reagent reservoir can be stored at room temperature.

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

Brief Protocol
  • 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 as 25 ng or as much as 5 g 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 RNA yield greater number of positive calls; that is, genes expressed in the linear dynamic range of the method.

  • Important: Use a consistent amount of total RNA for all samples in a single experiment to be characterized and compared.
  1. Remove the tubes of GE2 Solution and BC4 Solution from -20ºC storage and thaw on ice
  2. Briefly centrifuge the tubes after thawing. Keep the tubes on ice until they are used.
  3. Transfer 750 µl of the GE2 Solution to one of the included 5-ml reagent reservoirs. For best results, use a pipette to transfer the GE2 Solution into the reagent reservoir. Decanting the GE2 Solution from the tube into the reservoir is not recommended.
  4. Add 6 l of GE2 Solution from the reagent reservoir to each well of the empty 96-well plate with a pipette.
  5. Add 8 l of a RNA sample to each well with a pipette and mix by pipetting up and down. Completely seal the plate with the foil adhesive sealing film.
    Note: The amount of total RNA per well should be consistent.
  6. Centrifuge the plate at 1000 g for 1 minute.
  7. Incubate plate with compression mat on top at 37ºC for 5 minutes in a thermal cycler (or room temperature for 10 minutes).
  8. Carefully remove the foil seal.
  9. Transfer 750 l of the BC4 Solution (RT Master Mix) to the other included 5-ml reagent reservoir. For best results, use a pipette to transfer the BC4 Solution into the reagent reservoir. Decanting the BC4 Solution from the tube into the reservoir is not recommended.
  10. Transfer 6 l of BC4 (RT Master Mix) from the reagent reservoir to each well of the 96-well plate, and mix by pipetting up and down. Completely seal the plate with a new piece of foil adhesive sealing film.
  11. Centrifuge the plate at 1000 g for 1 minute.
  12. Perform the reverse transcription (this step may be performed in any thermal cycler)
    1. Set up a program for 42ºC 15 minutes, 95ºC 5 minutes, 4ºC hold (forever)
    2. Put the plate in the thermal cycler with the reusable compression mat on top of the plate.
    3. Close lid of the thermal cycler, and run the program.
  13. Keep the plate with the finished reaction on ice until ready to use for real-time PCR, or place the plate at -20ºC for long-term storage.
  14. If performing analysis using RT² Profiler PCR Arrays or Primer Assays,
    1. Thaw plate, and then centrifuge the plate at 1000 g for 1 minute.
    2. Transfer the cDNA to new tubes and add 91 l H2O.
    3. Continue with protocol in the PCR Array User Manual or PCR Primer Assay User Manual.