Taq or phusion

Total RNA was isolated from dark-adapted zebrafish retinae of control, injured, and drug-treated/MO-electroporated/mRNA-transfected groups using TRIzol (Invitrogen). A combination of oligo-dT and random hexamers were used to reverse-transcribe ∼5 μg of RNA using Superscript III Reverse Transcriptase (Invitrogen) to generate cDNA. PCR reactions used Taq or Phusion (New England Biolabs) polymerase and gene-specific primers (Table S1) with previously described cycling conditions (Ramachandran et al, 2010a (link)). Quantitative real-time PCR (qRT-PCR) was carried out in triplicate with KOD SYBR (SYBR green containing PCR mix with KOD DNA polymerase from Thermococcus kodakaraensis) qRT-PCR mix (QKD-201; Genetix) on a real-time PCR detection system (MasterCycler RealPlex4; Eppendorf). The let-7a miRNA levels were determined with TaqMan hsa-let7-a probe (Applied Biosystems) as per the manufacturer’s instructions. The relative expression of mRNAs in control and injured retinae was deciphered using the ΔΔCt method and normalized to β-actin mRNA levels.

Total RNA was isolated from dark-adapted zebrafish retinae of control, injured, and drug-treated/MO-electroporated group using TRIzol (Invitrogen). Combination of oligo-dT and random hexamers were used to reverse transcribe 5 µg of RNA using Superscript II reverse transcription (Invitrogen) to generate cDNA. PCR reactions used Taq or Phusion (New England Biolabs) DNA polymerase and gene-specific primers (Table S1) with previously described cycling conditions (Ramachandran et al., 2010a (link)). qPCR was performed in triplicate with KOD SYBR qPCR mix (QKD-201; Genetix) as per manufacturer’s recommendations on a real-time PCR detection system (Eppendorf Master Cycler RealPlex4). The relative expression of mRNAs in control and injured retinae was deciphered using the ΔΔCt method and normalized to ribosomal protein l-24 or β-actin mRNA levels.