Lunascript rt supermix kit

HSC LX-2 cells were lysed by addition of 1 ml TRIzol™ and
incubation at room temperature for 5 min. Phase separation was performed by
addition of 100 μl 1-bromo-3-chloropropane and centrifugation at
12,000 ×g and 4 °C for 15 min. Supernatant
was transferred and total RNA was precipitated by addition of 500 μl
2-propanol and centrifugation at 12,000 ×g and 4
°C for 10 min. RNA was washed twice with 70% ethanol and
centrifugation at 7500 ×g and 4 °C for 5 min.
DNA digestion was performed with DNase I and RNA was reverse-transcribed
into cDNA using LunaScriptTM RT SuperMix kit (New England Biolabs). qPCR was
performed as described previously [27 (link)]. The following primers were used: PNPLA2_forward: 5′-GTG
TCA GAC GGC GAG AAT G -3′; PNPLA2_reverse: 5′- TGG AGG GAG GGA
GGG ATG -3′; LIPE_forward: 5′- CTG CAT AAG GGA TGC TTC TAT GG
-3′; LIPE_reverse: 5′- GCC TGT CTC GTT GCG TTT G -3′;
PNPLA3_forward: 5′- GGC ATC TCT CTT ACC AGA GTG T -3′;
PNPLA3_reverse: 5′- GGC ATC CAC GAC TTC GTC TTT -3′;
ABHD5_-forward: 5′- ACA GAC CTG TCT ATG CTT TTG AC -3′;
ABHD5_reverse: 5′- AGG GCA CAT CTC CAC TCT TCA -3′;
36B4_forward: 5′- GCT TCA TTG TGG GAG CAG ACA -3′;
36B4_reverse: 5`- CAT GGT GTT CTT GCC CAT CAG-3′. Target gene
expression was calculated by the ΔΔCT method. Expression of
ribosomal housekeeping gene 36b4 was used for
normalization.