Input Requirements

Total RNA Input

  • The protocol is optimized for 10–100 ng of human total RNA.
    • Lower amounts might result in low yield and reduced sensitivity.
    • Use NanoDrop to determine FFPE RNA concentration.
  • The protocol has been tested using 10 ng of high-quality universal human reference total RNA as input and 20–100 ng of total RNA extracted from FFPE.
    • Use of RNA from other tissues or lower quality RNA might require further optimization to determine the input amount.
  • For FFPE or degraded samples, determine the quality of the RNA starting material, then calculate the DV200 to determine input.
    • Use the Agilent RNA 6000 Nano Kit or Advanced Analytical Standard Sensitivity RNA Analysis Kit to determine the quality of your starting material.
  • Degraded or FFPE RNAs are shorter than full length RNA.
    • DNA contamination causes an underestimation of the amount of RNA used.
    • If starting with FFPE RNA, the sample input amount is based on sample quality. Use the percentage of RNA fragments > 200 nt fragment distribution value (DV200) as a reliable determinant of FFPE RNA quality.
    • The protocol has been tested using 20–100 ng of total RNA extracted from FFPE as input. The following input recommendations for FFPE RNA (20–100 ng) are provided as a guide for sample-specific optimization.

Quality

DV200

Input Requirement Per Reaction

High

>70%

20 ng

Medium

50-70%

20-50 ng

Low

30-50%

50-100 ng

Too degraded

< 30%

Not recommended

  • For successful library prep, use an RNA isolation method that includes a reverse-crosslinking step and DNase1 treatment, such as the QIAGEN RNeasy FFPE Kit or QIAGEN AllPrep DNA/RNA FFPE Kit.
  • For samples that border a quality classification, err towards the higher end of the input recommendation.
  • Poor results may be obtained with low quality FFPE samples.

Control

Use Agilent Technologies Human UHR total RNA (catalog # 740000) as a control sample for this protocol.