For detection of piRNAs in zucchini(RNAi) samples, total RNA was run on a 12% denaturing acrylamide gel and stained with SYBR gold for 30 min at room temperature. Analysis of RNA from smedwi-1(RNAi) and control neoblasts was performed by radioactive labeling. RNA was end-labeled with 32P, separated on a 12% denaturing acrylamide gel, vacuum dried, and imaged using a phosphorimager screen on a GE Typhoon FLA 9000 gel imager.
RNase protection assay was performed using an internally labeled ssRNA probe purified by size selection from gel. The probe was hybridized with the RNA sample overnight at 46°C, followed by digestion with RNaseA (40ug per sample), RNaseT1 (1U), and RNase One (20U) for 45 min at 40°C, followed by 30 min at 37°C. RNases were inactivated by Proteinase K digestion. Protected RNA was purified by TRIzol extraction, separated on a 12% denaturing acrylamide gel, vacuum dried, and imaged using a phosphorimager screen on a GE Typhoon FLA 9000 gel imager.
RNase protection assay was performed using an internally labeled ssRNA probe purified by size selection from gel. The probe was hybridized with the RNA sample overnight at 46°C, followed by digestion with RNaseA (40ug per sample), RNaseT1 (1U), and RNase One (20U) for 45 min at 40°C, followed by 30 min at 37°C. RNases were inactivated by Proteinase K digestion. Protected RNA was purified by TRIzol extraction, separated on a 12% denaturing acrylamide gel, vacuum dried, and imaged using a phosphorimager screen on a GE Typhoon FLA 9000 gel imager.