Rn_Rab7_5 FlexiTube siRNA(r)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Neonatal rats at postnatal day 0 to day 2 were sacrificed by decapitation and subsequent heart removal. Ventricular myocytes were then isolated using the Cellutron Neomyocytes isolation system (Cellutron Life Technology, Baltimore, MD) following the manufacturer's instructions [22]. Cells collected were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µM BrdU, and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). To selectively enrich cardiomyocytes, the cells were preplated in 100 mm non-coated dishes for 1 hour. The resulting suspended cells were counted with a hemocytometer and then plated evenly on 1% gelatin-coated plates in appropriate cell densities. The plated cells were then cultured in a 5% CO2 incubator at 37°C for at least 24 hours before the medium was changed to meet the needs of the follow-up experiments. The siRNA's specific for Rab7 (siRab7: Cat. # SI03021725) and the siRNA targeting luciferase serving as a control siRNA (siLuc: 5′-AACGTACGCGGAATACTTCGA-3′) were all purchased from Qiagen (Valencia, CA). LipofectamineTM 2000 transfection reagent (Invitrogen) was used for siRNA transfection following the manufacturer's protocol. Transfection of cultured NRVMs with siRNA was generally started at 48–72 hours after NRVMs were plated . Six hours after the transfection, the siRNA-containing medium was replaced with the fresh medium containing 2% FBS. . For the Rab7 knockdown experiments, Rab7 siRNA were introduced along the second round of sip62 or siLuc transfection

Protocol tips

Neonatal rats at postnatal day 0 to day 2 were sacrificed by decapitation and subsequent heart removal. Ventricular myocytes were then isolated using the Cellutron Neomyocytes isolation system (Cellutron Life Technology, Baltimore, MD) following the manufacturer's instructions [22]. Cells collected were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µM BrdU, and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). To selectively enrich cardiomyocytes, the cells were preplated in 100 mm non-coated dishes for 1 hour. The resulting suspended cells were counted with a hemocytometer and then plated evenly on 1% gelatin-coated plates in appropriate cell densities. The plated cells were then cultured in a 5% CO2 incubator at 37°C for at least 24 hours before the medium was changed to meet the needs of the follow-up experiments. The siRNA's specific for Rab7 (siRab7: Cat. # SI03021725) and the siRNA targeting luciferase serving as a control siRNA (siLuc: 5′-AACGTACGCGGAATACTTCGA-3′) were all purchased from Qiagen (Valencia, CA). LipofectamineTM 2000 transfection reagent (Invitrogen) was used for siRNA transfection following the manufacturer's protocol. Transfection of cultured NRVMs with siRNA was generally started at 48–72 hours after NRVMs were plated . Six hours after the transfection, the siRNA-containing medium was replaced with the fresh medium containing 2% FBS. . For the Rab7 knockdown experiments, Rab7 siRNA were introduced along the second round of sip62 or siLuc transfection

Publication protocol

Neonatal rats at postnatal day 0 to day 2 were sacrificed by decapitation and subsequent heart removal. Ventricular myocytes were then isolated using the Cellutron Neomyocytes isolation system (Cellutron Life Technology, Baltimore, MD) following the manufacturer's instructions [22]. Cells collected were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µM BrdU, and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). To selectively enrich cardiomyocytes, the cells were preplated in 100 mm non-coated dishes for 1 hour. The resulting suspended cells were counted with a hemocytometer and then plated evenly on 1% gelatin-coated plates in appropriate cell densities. The plated cells were then cultured in a 5% CO2 incubator at 37°C for at least 24 hours before the medium was changed to meet the needs of the follow-up experiments. The siRNA's specific for Rab7 (siRab7: Cat. # SI03021725) and the siRNA targeting luciferase serving as a control siRNA (siLuc: 5′-AACGTACGCGGAATACTTCGA-3′) were all purchased from Qiagen (Valencia, CA). LipofectamineTM 2000 transfection reagent (Invitrogen) was used for siRNA transfection following the manufacturer's protocol. Transfection of cultured NRVMs with siRNA was generally started at 48–72 hours after NRVMs were plated . Six hours after the transfection, the siRNA-containing medium was replaced with the fresh medium containing 2% FBS. . For the Rab7 knockdown experiments, Rab7 siRNA were introduced along the second round of sip62 or siLuc transfection

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing siRNA / miRNA gene silencing Rat - NRVM( Rab7 using Rn_Rab7_5 FlexiTube siRNA(r) from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for Rn_Rab7_5 FlexiTube siRNA(r) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing siRNA / miRNA gene silencing Rat - NRVM( Rab7 using Rn_Rab7_5 FlexiTube siRNA(r) from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.