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Your Data: Synthesizing Control RNAs for Real-Time RT-PCR Viral Quantification

Dr. Luke T Daum and colleagues (Longhorn Vaccines & Diagnostics, San Antonio, Texas) use real-time RT-PCR to quantify viral copies present in nose and lung homogenates from cotton rats (S. hispidus) challenged with influenza A virus. This article describes how they made the experimental controls using Ambion® kits and reagents.

Luke T Daum¹, James P Chambers¹, ², Jeff Fischer¹, and Gerald W Fischer^1
1Longhorn Vaccines & Diagnostics
San Antonio, TX 78209
²The University of Texas at San Antonio
San Antonio, TX 78249

Relative Quantitation and Controls
Relative Quantitation (RQ; also called Comparative C_T) is an established method for using real-time RT-PCR to measure change in levels of a target nucleic acid sequence (e.g., influenza A) relative to the same sequence in a known calibrator sample. The exogenous control is a distinct nucleic acid sequence (e.g., influenza B) of known concentration added to each sample and serves as a reference to control for variations from extraction and pipetting. For Dr. Daum's experiments, calibrator and exogenous control complementary RNA (cRNA) were synthesized for normalizing viral concentration and controlling for sample variation, respectively.

Synthesis of Controls
To make the control cRNA, Dr. Daum and colleagues used RT-PCR to incorporate an overhanging 5' T7 promoter sequence into influenza A and B amplicons. The MEGAscript® T7 Transcription Kit was then used to generate RNA transcripts from these amplicons. The resulting RNA was treated with TURBO DNase™ enzyme to destroy template DNA, and purified using the MEGAclear™ Kit to remove salts, buffers, primers, and enzymes. The calibrator and exogenous control RNA were stored in THE RNA Storage Solution and used as reliable and consistent calibrator and exogenous control template RNA for quantifying influenza A virus from cotton rat nose and lung homogenates (Figure 1). Real-time RT-PCR was performed using RQ with the Applied Biosystems 7500 Real-Time PCR System.

Figure 1. Real-Time RT-PCR Assays Detect Known Amounts of Calibrator and Exogenous Control Template. The calibrator and exogenous control reactions represent 0.1 pg of template cRNA corresponding to approximately 5.8 x 105 influenza copies. The calibrator and exogenous control were used in subsequent real-time RT-PCR to quantify viral copy number from cotton rat nose and lung samples infected with influenza A virus.

For Research Use Only. Not for use in diagnostic procedures.