Fig. 4.
Fig. 4. RT-PCR accurately detect BCR/ABL mRNA levels. (A) Presence of AS mRNA does not impede RT-PCR reaction for BCR/ABL mRNA. mRNA from untransduced 32Dp210 cells was admixed with 0 (lane A), onefold (lane B), fivefold (lane C), or 10-fold (lane D) excess mRNA from LasBD-PA317 cells immediately before the RT-PCR reaction. After reverse transcription with MoMLVRT, cDNAs were amplified for 30 cycles. The addition of up to 10-fold excess LasBD-PA317 mRNA containing the double-copy AS mRNA did not decrease our ability to assess the level of BCR/ABL mRNA in 32Dp210 cells. (B) RT-PCR using Tth or MoMLVRT/Taq polymerase. RT-PCR reaction was performed either at 68°C for 20 minutes using the Tth polymerase or at 37°C for 60 minutes using MoMLVRT/Taq polymerase. mRNA obtained from LasBD-32Dp210 clone A and bulk-selected LasBD-32Dp210 cells was amplified for 30 cycles, and the degree of BCR/ABL mRNA suppression detected using either MoMLVRT or Tth was similar.

RT-PCR accurately detect BCR/ABL mRNA levels. (A) Presence of AS mRNA does not impede RT-PCR reaction for BCR/ABL mRNA. mRNA from untransduced 32Dp210 cells was admixed with 0 (lane A), onefold (lane B), fivefold (lane C), or 10-fold (lane D) excess mRNA from LasBD-PA317 cells immediately before the RT-PCR reaction. After reverse transcription with MoMLVRT, cDNAs were amplified for 30 cycles. The addition of up to 10-fold excess LasBD-PA317 mRNA containing the double-copy AS mRNA did not decrease our ability to assess the level of BCR/ABL mRNA in 32Dp210 cells. (B) RT-PCR using Tth or MoMLVRT/Taq polymerase. RT-PCR reaction was performed either at 68°C for 20 minutes using the Tth polymerase or at 37°C for 60 minutes using MoMLVRT/Taq polymerase. mRNA obtained from LasBD-32Dp210 clone A and bulk-selected LasBD-32Dp210 cells was amplified for 30 cycles, and the degree of BCR/ABL mRNA suppression detected using either MoMLVRT or Tth was similar.

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