Total RNA was purified from [A] 102–107 Jurkat cells using the miRNeasy 96 Kit or [B] a dilution series of rat lung tissue homogenate from 20 mg to 200 ng using the miRNeasy Mini Kit. miRNA-enriched fractions (<200 nt) were also isolated from the same samples. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16.
Total RNA including miRNA was purified from 25 mg of a range of RNAlater stabilized rat tissues using the miRNeasy 96 Kit. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16 and for the larger mRNA of the PGK1 gene. Results showed successful detection of both PGK1 mRNA (large RNA) and miR-16 (small RNA) from the same eluates.
Total RNA including miRNA was purified from a range of amounts of Jurkat cells using the miRNeasy Mini Kit or TRIzol Reagent. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16. Results showed that CT values were lower after purification using the miRNeasy Kit, indicating that higher amounts of miRNA were purified than using TRIzol. miRNA was effectively purified from as little as 1 x 102 cells using the miRNeasy Kit. In contrast, no miRNA was detected after 40 PCR cycles from 1 x 102 cells when TRIzol was used for purification.
miRNA-enriched fractions were purified from 3 x 106 HeLa cells using either the miRNeasy Mini Kit or a kit from Supplier AIV. Equal RNA amounts from miRNA-enriched fractions were run on a 1.5% gel. For comparison, 20mer and 21mer synthetic RNAs were also run on the gel. The miRNA-enriched fraction prepared using the miRNeasy Kit showed a smear of RNAs of different sizes down to approximately 18 nt. In contrast, in the miRNA-enriched fraction prepared using the kit from Supplier AIV, primarily tRNA and 5S rRNA were visible.
Total RNA including miRNA was purified from 1 x 106 Jurkat cells using the miRNeasy Mini Kit or TRIzol Reagent. The absorbance spectra showed the OD maximum for RNA purified using the miRNeasy Kit was at 260 nm. In contrast, the OD maximum was greater than 260 nm when TRIzol was used for purification, indicating phenol carryover. In addition, the OD230 measurement was higher in the TRIzol-prepared RNA, indicating salt carryover from the TRIzol Reagent.
The miRNeasy Mini Kit enables purification of total RNA which includes RNA from 18 nucleotides (nt) upwards. Alternatively, an miRNA-enriched fraction and a total RNA (>200 nt) fraction can be can be purified separately. A separate miRNA-enriched fraction may be required to reduce background in particularly sensitive downstream applications.