These specific samples were processed with the SMART-Seq HT kit as described in the user manual, including one-step RT-PCR and amplified with 17 cycles of PCR. Example electropherogram traces from Agilent 2100 Bioanalyzer. These traces are representative of results obtained from SMART-Seq HT and SMART-Seq v4 kits. Unfortunately, we have found that the PerkinElmer LabChip system is not sensitive enough for analysis of the cDNA produced by the SMART-Seq v4 or SMART-Seq HT kits.įigure 1. The Fragment Analyzer generally gives results similar to the Bioanalyzer, though the same samples may look different when analyzed with the Bioanalyzer versus the Agilent 2200 TapeStation (Figure 2). These values and shapes may differ if you overload your chip or use an alternative capillary electrophoresis instrument. # P11496), the yield in your negative control should be ≤100 pg/µl. If using a fluorescent nucleic acid binding dye such as PicoGreen (Thermo Fisher Scientific, Cat. For the negative control, cDNA synthesis and amplification should yield very little to no product using the Agilent High Sensitivity DNA Kit (Figure 1, Panel C). The cDNA should show a distinct peak that spans 400 to 10,000 bp and has a maximum between 2,000 and 2,500 bp (Figure 1, Panel A) when analyzed with an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit (Agilent, Cat. Successful cDNA synthesis and amplification in the positive control RNA sample should yield ≥ 200 pg/µl of cDNA. SMART-Seq v4 and SMART-Seq HT: expected results As different kits produce different results, please go to the appropriate section for SMART-Seq HT, SMART-Seq v4, or SMART-Seq Stranded. This article can help explain how these controls should perform and help you troubleshoot when they do not. New users can sometimes also see a high background in their negative controls, which is a critical problem. Until you are comfortable with the protocol, you may want to test two positive control inputs (e.g., 10 pg and 100 pg). Due to the difficulties of working with very small input masses, some new users may see low cDNA yield in their positive control samples. Similarly, the best negative control is one treated the same as your actual samples (e.g., mock FACS sample buffer). The best positive control has an RNA input mass similar to your experimental samples (e.g., 10 pg of RNA as a starting point for single cells). All of Takara Bio's NGS kits include positive control samples to make this as easy as possible. Whether you've done single-cell RNA-seq one or one thousand times before, control reactions are invaluable for troubleshooting your experiments.
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