Quantitative-PCR protocol for A. mexicanus
Brain tissues will be recovered from
· 1 month old (5 individuals from each cave or surface population)
· 2 month old (5 individuals from each cave or surface population)
· 4 month old (3 individuals from each cave or surface population)
· 12 month old (3 individuals from each cave or surface population)
Dissected in ice-cold PBS, and quickly recovered in 1.5 mL tubes on dry-ice (quick freeze method). Each different stage (4 stage) from each population (2 populations: Pachón cavefish and surface fish) is collected in a tube so it will be 8 of 1.5 mL tubes
Brain tissues are suspended with 400 µL of QIAzol Lysis Reagent, and are grinded by disposable molar thoroughly.
Add 500 µL of QIAzol Lysis Reagent to make it total 900 µL.
Follow the protocol of QIAGEN RNeasy Plus Universal Mini kit.
Elute total RNA with 2 x 50 µL (100 µL in total) of RNase-fee water.
Quantify RNA amount with ThermoFisher Scientific Qubit 3.0
Store in -80˚C
Use 1µg of total RNA for cDNA synthesis.
Follow the protocol of Bio-Rad iScriptTM gDNA Clear cDNA Synthesis Kit
Quantify single strand DNA amount (cDNA) with ThermoFisher Scientific Qubit 3.0
Store in -20˚C
Design 2 set of primers per gene by using perlprimer software (http://perlprimer.sourceforge.net/). These primers should be designed to target different exon-intron boundaries.
Run the qPCR by using 10ng of adult brain cDNA (surface fish and cavefish) per 10µL reaction using 56˚C as annealing temperature. Follow the protocol of SsoAdvanced™ Universal SYBR® Green. After 40 cycles of qPCR, run the melting-curve analysis. These qPCR is conducted with Bio-Rad CFX96 qPCR machine.
With melting curve result, select one pair of primers per gene
Using 1ng of brain cDNA of each sample (4 developmental stages x 2 populations), qPCR will be conducted with each primer set. In addition to these target genes, three reference house-keeping genes (b2m, rps18 and eef2a.1) will be tested in the same plate. Each data-point is triplicated, and in later, averaged.