Omniscript rt kit

The RNA samples for RT-PCR were prepared from logarithmically growing D. discoideum cells washed in 17 mM phosphate buffer (pH 6.2) and stored as pellets of 2 × 10⁷ cells at –80°C until further use. Total RNA was prepared from frozen cells using the Qiagen RNeasy^® Mini Kit according to the provided manual. cDNA was synthesized by the reverse transcription of 500 ng of total RNA using the Qiagen Omniscript^® RT Kit. RT-PCR on circularized RNA (cRT-PCR) was performed as previously described [29 (link)]. Briefly, the total RNA was 5'-dephosphorylated with shrimp alkaline phosphatase (Fermentas) and then re-phosphorylated using Fermentas T4 polynucleotide kinase. The phosphorylated RNA was then self-ligated using T4 RNA ligase (Fermentas). Reverse transcription was performed using the primer cRT-PCR-01 and the Qiagen Omniscript^® RT Kit. The first PCR was conducted with the primers cRT-PCR-02 and cRT-PCR-03 using the following cycling conditions: 95°C for 5 min and 35 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 45 sec. The PCR products were purified using Qiagen's PCR Purification Kit, followed by a nested PCR using the same cycling conditions and the primers cRT-PCR-04 and cRT-PCR-05. The sequences of the cRT-PCR primers for each locus are listed in (S1 Table). The cRT-PCR products were analyzed on agarose gels, cloned into pGEM-T (Promega) and sequenced.

The 21-gene tests were performed on formalin-fixed, paraffin-embedded tissue. Hematoxylin and eosin-stained slides were deparaffinized into two 10-µm unstained sections using xylene followed by ethanol as we described in our previous study (15 (link)). RNA was extracted and purified using the RNeasy FFPE kit (QIAGEN, Hilden, Germany). Gene-specific reverse transcription was conducted using Omniscript RT kit (Qiagen, 205111, Germany). Standardized quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in 96-well plates with Applied Biosystems (Foster City, CA, USA) 7500 Real-Time PCR system. RT-PCR was carried out with the Omniscript RT kit (Qiagen, Valencia, CA, USA). Expression of each gene was measured in triplicate, and normalized relative to a set of five reference genes.

Total RNA was extracted with Trizol (Invitrogen). After reverse-transcription with Omniscript RT Kits (Qiagen), cDNA was amplified by primers for Hes1, 5′- GTGGGTCCTAACGCAGTGTC-3′ and R5′-GTCAGAAGAGAGAGGTGGGCTA-3′; For real-time PCR, after reverse-transcription with Omniscript RT Kits (Qiagen), SYBR premix Ex Taq II (Takara Bio) was used for quantitative PCR. All data were normalized to HPRT (hypoxanthine-guanine phosphoribosyl transferase) and were presented as fold increase relative to the background value. The sequences for primers were as follows: 5′-CACGGCCCTGGTTCTCAT-3′; reverse primer, 5′-CAGATGTTCCACTCTCCTCTTCTCT-3′, Actin, 5′-TGGGAATGGGTCAGAAGGACT-3′ and 5′-TTTCACGGTTGGCCTTAGGGT-3′