After extracting the total RNA using Gen Elute Mammalian Total RNA
miniprep kits (Sigma), and checking its integrity by electrophoresis, the
cDNA was synthesized from 1 mg of purified total RNA using Revert Aid H
minus first strand cDNA synthesis kit (Fermentas Life Sciences,
Ontario,Canada). Expression of mouse and human IPMK, mouse LC3B, mouse BNIP3
and mouse GAPDH was detected using suitably designed Taqman primers
(Invitrogen).Other genes such as mouse BNIP3L, P62,GABARAPL1 and ATG12 were
determined using primers given in the table. qPCR of these genes was
performed using power Sybr green pcr mater mix from Invitrogen. Expression
of the designated enzymes was normalized against glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as the internal reference. The experiments were
performed (real-time PCR Systems StepOne plus, Applied Biosystems) in
triplicate. Data were quantified for the above genes using the comparative
Ct method, as described in the Assays-on-Demand Users Manual (Applied
Biosystems).
qPCR Primers (Sybr green):
catcgtggagaaggctccta- gabarapl1 (mF)
atacagctggcccatggtag- gabarapl1 (mR)
AACAAAGAAATGGGCTGTGG – ATG12 (mF)
TTGCAGTAATGCAGGACCAG- ATG12 (mR)
CCTCGTCTTCCATCCACAAT- bnip3l (mF)
GTCCCTGCTGGTATGCATCT- bnip3l (mR)
TGGCCACCTCTCTGATAGCT- p62 (mF)
TCATCGTCTCCTCCTGAGCA- p62 (mR)
miniprep kits (Sigma), and checking its integrity by electrophoresis, the
cDNA was synthesized from 1 mg of purified total RNA using Revert Aid H
minus first strand cDNA synthesis kit (Fermentas Life Sciences,
Ontario,Canada). Expression of mouse and human IPMK, mouse LC3B, mouse BNIP3
and mouse GAPDH was detected using suitably designed Taqman primers
(Invitrogen).Other genes such as mouse BNIP3L, P62,GABARAPL1 and ATG12 were
determined using primers given in the table. qPCR of these genes was
performed using power Sybr green pcr mater mix from Invitrogen. Expression
of the designated enzymes was normalized against glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as the internal reference. The experiments were
performed (real-time PCR Systems StepOne plus, Applied Biosystems) in
triplicate. Data were quantified for the above genes using the comparative
Ct method, as described in the Assays-on-Demand Users Manual (Applied
Biosystems).
qPCR Primers (Sybr green):
catcgtggagaaggctccta- gabarapl1 (mF)
atacagctggcccatggtag- gabarapl1 (mR)
AACAAAGAAATGGGCTGTGG – ATG12 (mF)
TTGCAGTAATGCAGGACCAG- ATG12 (mR)
CCTCGTCTTCCATCCACAAT- bnip3l (mF)
GTCCCTGCTGGTATGCATCT- bnip3l (mR)
TGGCCACCTCTCTGATAGCT- p62 (mF)
TCATCGTCTCCTCCTGAGCA- p62 (mR)