heat shock protein family A (Hsp70) member 5

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	PC3 (bone metastatic PCa cell line, ATCC®-CRL-1435), was purchased from ATCC. PC3 cells were cultured in RPMI-1640 medium containing 10% FBS, 2.5 mM of L-glutamine and 1% penicillin/streptomycin. Both cell lines were cultured at 37 °C in a humidified tissue culture incubator containing 5% CO2. Cells were maintained and propagated at 33 °C and 5% CO2 except during co-culture experiments which were conducted at 37 °C. All cell lines were periodically checked for mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza). Authentication of cell lines was performed by STR DNA profiling analysis conducted by the Molecular Resources Facility at Rutgers University. Cell populations were frozen after 3 passages from the time of initial receipt and growth and were discarded after 30 passages.HSPA5 (GRP78) targeting siRNAs were purchased from Ambion (Carlsbad, USA). Two different siRNAs; Silencer® Select Pre-designed siRNA s6979 (5’ UUC UGG ACG GGC UUC AUA Gtt 3′) and s6980 (5’ UCU AGU AUC AAU GCG CUC Ctt 3′) targeting exons 6 and 8, respectively, were tested. For control, the Silencer® select negative control No. 2 siRNA was used (Ambion). siRNA transfections were performed using a modified reverse transfection technique [32] using a cocktail containing equimolar quantities of each GRP78 siRNA to maximize silencing potential. The GRP78 siRNA cocktail (or siRNA control) was diluted in Opti-MEM reduced serum medium and incubated with the TransIT-X2 dynamic delivery system (Mirus Bio) according to the manufacturer’s protocol. The siRNA-TransIT-X2 complexes were added to wells of either a 6- or 24- well plate upon which PC3 cells seeded in complete growth medium at a cell density of 7.5-9 × 105 cells/well (6 well plate) or 0.75-1 × 105 cells/well (24 well plate). GRP78 siRNA cocktail or control siRNA were used at a final concentration of 50 nM for PC3 cell line.

Protocol tips

PC3 (bone metastatic PCa cell line, ATCC®-CRL-1435), was purchased from ATCC. PC3 cells were cultured in RPMI-1640 medium containing 10% FBS, 2.5 mM of L-glutamine and 1% penicillin/streptomycin. Both cell lines were cultured at 37 °C in a humidified tissue culture incubator containing 5% CO2. Cells were maintained and propagated at 33 °C and 5% CO2 except during co-culture experiments which were conducted at 37 °C. All cell lines were periodically checked for mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza). Authentication of cell lines was performed by STR DNA profiling analysis conducted by the Molecular Resources Facility at Rutgers University. Cell populations were frozen after 3 passages from the time of initial receipt and growth and were discarded after 30 passages.HSPA5 (GRP78) targeting siRNAs were purchased from Ambion (Carlsbad, USA). Two different siRNAs; Silencer® Select Pre-designed siRNA s6979 (5’ UUC UGG ACG GGC UUC AUA Gtt 3′) and s6980 (5’ UCU AGU AUC AAU GCG CUC Ctt 3′) targeting exons 6 and 8, respectively, were tested. For control, the Silencer® select negative control No. 2 siRNA was used (Ambion). siRNA transfections were performed using a modified reverse transfection technique [32] using a cocktail containing equimolar quantities of each GRP78 siRNA to maximize silencing potential. The GRP78 siRNA cocktail (or siRNA control) was diluted in Opti-MEM reduced serum medium and incubated with the TransIT-X2 dynamic delivery system (Mirus Bio) according to the manufacturer’s protocol. The siRNA-TransIT-X2 complexes were added to wells of either a 6- or 24- well plate upon which PC3 cells seeded in complete growth medium at a cell density of 7.5-9 × 105 cells/well (6 well plate) or 0.75-1 × 105 cells/well (24 well plate). GRP78 siRNA cocktail or control siRNA were used at a final concentration of 50 nM for PC3 cell line.

Publication protocol

PC3 (bone metastatic PCa cell line, ATCC®-CRL-1435), was purchased from ATCC. PC3 cells were cultured in RPMI-1640 medium containing 10% FBS, 2.5 mM of L-glutamine and 1% penicillin/streptomycin. Both cell lines were cultured at 37 °C in a humidified tissue culture incubator containing 5% CO2. Cells were maintained and propagated at 33 °C and 5% CO2 except during co-culture experiments which were conducted at 37 °C. All cell lines were periodically checked for mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza). Authentication of cell lines was performed by STR DNA profiling analysis conducted by the Molecular Resources Facility at Rutgers University. Cell populations were frozen after 3 passages from the time of initial receipt and growth and were discarded after 30 passages.HSPA5 (GRP78) targeting siRNAs were purchased from Ambion (Carlsbad, USA). Two different siRNAs; Silencer® Select Pre-designed siRNA s6979 (5’ UUC UGG ACG GGC UUC AUA Gtt 3′) and s6980 (5’ UCU AGU AUC AAU GCG CUC Ctt 3′) targeting exons 6 and 8, respectively, were tested. For control, the Silencer® select negative control No. 2 siRNA was used (Ambion). siRNA transfections were performed using a modified reverse transfection technique [32] using a cocktail containing equimolar quantities of each GRP78 siRNA to maximize silencing potential. The GRP78 siRNA cocktail (or siRNA control) was diluted in Opti-MEM reduced serum medium and incubated with the TransIT-X2 dynamic delivery system (Mirus Bio) according to the manufacturer’s protocol. The siRNA-TransIT-X2 complexes were added to wells of either a 6- or 24- well plate upon which PC3 cells seeded in complete growth medium at a cell density of 7.5-9 × 105 cells/well (6 well plate) or 0.75-1 × 105 cells/well (24 well plate). GRP78 siRNA cocktail or control siRNA were used at a final concentration of 50 nM for PC3 cell line.

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 Human - PC3 (human prostate cancer cell line) HSPA5 (GRP78) using heat shock protein family A (Hsp70) member 5 from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for heat shock protein family A (Hsp70) member 5 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) HSPA5 (GRP78) using heat shock protein family A (Hsp70) member 5 from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.