reverse transcription PCR (RT-PCR) (Animalia)

Knowledge graph centered on reverse transcription PCR (RT-PCR) (Animalia) with 30 nodes and 107 connections. Top connected: Lepidoptera, Bacteria, Arabidopsis, Colias, chickens.

Method synopsis

A multi-step molecular protocol involving RNA extraction, reverse transcription, PCR amplification using consensus primers, TA cloning, and bidirectional Sanger sequencing to characterize genetic variants in the PEPCK gene.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — A genetic polymorphism evolving in parallel in two cell compartments and in two clades (2013), BMC Evolutionary Biology. Implementations in other papers (listed below) may differ.

1. Specimen collection
   Collected 18 Colias eurytheme individuals randomly sampled near Tracy, California (elevation 25 m) and 18 Colias meadii individuals randomly sampled from Cottonwood Pass, Colorado (elevation 3780 m). Fat body tissue was preserved in Ambion RNAlater for RNA extraction.
   Quantities: 36 total individuals: 18 C. eurytheme, 18 C. meadiiDuration: Not specifiedConditions: Field collection at two geographically distinct sites
   Equipment: Ambion RNAlater
2. RNA extraction and cDNA synthesis
   Extracted total RNA from fat body tissue using Invitrogen MiniV enzyme. Synthesized cDNA using reverse-transcription with random hexamer primers.
   Quantities: RNA from each of 36 individual specimensDuration: Not specifiedConditions: Laboratory conditions
   Equipment: Invitrogen MiniV enzyme
3. PCR primer design and amplification
   Designed consensus primers for initial PCR amplification using Invitrogen HiFi Platinum Taq and Stratagene Robo-cyclers. Used a two-fragment approach to amplify the complete PEPCK cDNA sequence. Primers were designed using Oligo 6 software targeting the 3' end with antisense primer matching cDNA polyA tail (GEN22(15)-A3end), then used Ambion RLM-RACE kit to amplify the 5' end.

Typical Equipment

• Ambion RNAlater
• Invitrogen MiniV enzyme
• Invitrogen HiFi Platinum Taq
• Stratagene Robo-cyclers
• Oligo 6 software
• Ambion RLM-RACE kit
• Qiagen kits
• Qiacube processing robot
• ABI BigDye 3.1 reagents
• ABI 377 sequencer
• BioEdit
• DnaSP 5.1
• TargetP server
• PHASE algorithm

Output Measurements

• DNA sequences
• genetic variants
• haplotypes

Papers Using This Protocol (1)

Species Studied With This Protocol