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Table 4 Comparison of methods used to measure A-to-I RNA editing

From: Antisense sequencing improves the accuracy and precision of A-to-I editing measurements using the peak height ratio method

Method # Steps Days Cost/Sample Cost/
3 Analysesa
Relative Cost Sites/
Analysis
Peak Height Ratio 6 (RNA isolation, RT, PCR, PCR purification, sequencing, analysis) 3 $11.87 $35.62 1 Several
Poisoned Primer Extension 7 (RNA isolation, RT, primer labeling, PCR, gel, imaging, analysis) 1-2 $120.63 Fb$123.40 32P $133.24 Fb$157.84 32P 15.0 Fb
17.7 32P
One
Restriction Digest 9 (RNA isolation, RT, PCR, purification, RE digestion, purification, gel, imaging, analysis) 2 $10.37 Fb
$123.40 32P
$31.10 Fb
$135.70 32P
3.50 Fb
15.2 32P
One
Ultra High Throughput Sequencing 9 (RNA isolation, RT, PCR(×2), purification(×2), hybridization, sequencing, analysis) 21-28 $1609.80 $4829.39 136 One to Severalc
Clone Counting 10 (RNA isolation, RT, PCR, PCR purification, cloning, transformation, colony screening, colony growth, plasmid purification, sequencing, analysis) 6 $24.48 $561.25 15.8 Several
  1. a = Cost/3 Analyses is simply not three-fold the Cost/Rep as primers can be labeled and used in multiple samples on a single day of experiments
  2. b = Fluorescent assay
  3. c = the ability to read several editing sites is determined by proximity of editing sites
  4. Costs are based on using the materials listed in Additional File 1. Additional costs were based on pGEM-T Cloning System II, PureYield Plasmid Purification System (Promega), restriction enzymes (PshAI and HpyCH4V, New England Biolabs), KinaseMax Kit, Alexa Fluor 488 and NucAway (Invitrogen). Data for Ultra High Throughput Sequencing is based on Abbas et al., 2010 and consists of a single lane 86 bp single-end read on a Genome Analyzer. The cost/sample is the cost of performing a single experiment from one biological sample. The cost/3 analyses is the cost for three replicates from one biological sample. Relative cost is for assessing the four editing sites of Dα6 reported herein. The price of shipping, primers, gels, standard markers, imaging equipment, software, and labor were not included in the cost