Rtase

Total RNA was isolated by using the RNA isoplus (TaKaRa Bio, Shiga, Japan) according to manufacturer’s instruction. The final pellet was air dried and dissolved into RNase-free DEPC solution (TaKaRa Bio, Shiga, Japan). The RNA concentration was measured with the Nano-drop (Thermo Fisher Scientific Inc., Waltham, MA). First strand cDNA synthesis was performed in RNase-free DEPC solution containing 2 μg total RNA and 10 pmol oligo dT at 70°C for 5min, followed by double-strand synthesis in 5X RT buffer (Invitrogen, Carlsbad, CA) with 8 mM dNTP (BIO BASIC INC., Ontario, Canada), 200 unit/μL RTase (Invitrogen, Carlsbad, CA) at 37°C for 60 min and at 72°C for 15 min.

Frozen organs were homogenized with 300 μL ice cold RNA isoplus (TaKaRa
Bio, Shiga, Japan). After chloroform extraction and isopropyl alcohol
precipitation, RNA was dissolved in 20 μL RNase-free DEPC (TaKaRa Bio,
Shiga, Japan) solution. The RNA concentrations were measured with the Nano-drop
(Thermo Fisher Scientific Inc., Waltham, MA, USA). First strand cDNA synthesis
was performed using 2 μg RNA, 10 pmol oligo dT and RNase-free DEPC
solution at 70° for 5min, followed by double-strand synthesis in
5× RT buffer (Invitrogen, Carlsbad, CA, USA) with 8 mM dNTP (BIO BASIC
INC., Ontario, Canada), 200 unit/μL RTase (Invitrogen, Carlsbad, CA, USA)
and RNase-free DEPC solution at 37° for 60 min and at 72° for 15
min. qRT-PCR was performed in a total volume of 20 μL buffer solution
containing 2 μL of template cDNA, 10 μL of SYBR Green (Roche,
Manheim, Germany), and 10 pmol of each primer. Primer pairs were as follows:
NUCB2 forward 5’-AAAACCTTGGCCTGTCTGAA-3’; reverse
5’-CATCGATAGGAACAGCTTCCA-3’ and GAPDH forward
5’-TTGATGGCAACAATCTCCAC-3’; reverse
5’-CGTCCCGTAGACAAAATGGT-3’ (BIONICS, Seoul, Korea). The optimal
temperature cycling protocol was determined to be 95° for 5 min followed
by 45 reaction cycles at 95° for 10 s, 60° for 10 s and 72°
for 10 s using the LightCycler^® 480 Real-time PCR System
(Roche, Manheim, Germany).

Samples of tissues were homogenized with RNA isoplus (TaKaRa Bio, Shiga, Japan).
After chloroform extraction and isopropyl alcohol precipitation, RNA has
dissolved in RNase-free DEPC (TaKaRa Bio) solution. The RNA concentrations were
measured with the Nano-drop (Thermo Fisher Scientific, Waltham, MA, USA).
First-strand cDNA synthesis was performed using the extracted RNA and oligo dT,
followed by the double-strand synthesis in RT buffer (Invitrogen, Carlsbad, CA,
USA) with dNTP (BIO BASIC, Markham, ON, Canada) and RTase (Invitrogen). qRT-PCR
was performed in a buffer solution containing template cDNA, SYBR Green
(Enzynomics, Daejeon, Korea), and each primer. Primer pairs (Bioneer, Korea)
were as follows; 18S (Forward 5’-GTCTGTGATGCCCTTAGATG-3’, Reverse
5’-AGCTTATGACCCGCACTTAC-3’), Fas (Forward
5’-CTGCGATTCTCCTGGCTGTGAA-3’, Reverse 5’-CAACAACCATAG
GCGATTTCTGG-3’), FasL (Forward 5’-TCCGTGAGTTCACCAACCAA-3’,
Reverse 5’-TGAGTGGGGGTTCCCTGTTA-3’), IL-6 (Forward
5’-ACCAGAGGAAATTTTCAA TA-3’, Reverse
5’-TGATGCACTTGCAGAAAACA-3’), IL-1β (Forward
5’-GGTCAAAGG TTTGGAAGCAG-3’, Reverse
5’-TGTGAAATGCCACCTTTTGA-3’). The optimum temperature cycling
protocol was determined to be 95°C for 10 s, 60°C for 10 s, and
72°C for 10 s using the Light Cycler 480 Real-time PCR System (Roche,
Manheim, Germany).