Sensifasat sybr no rox kit

Total RNAs were extracted from whole flies (10♀) using Total RNA Purification Plus Kit (Norgen – Biotek) and cDNA was prepared from 0.5 µg total RNA using Maxima First Strand cDNA Syntesis Kit (ThermoScientific). Triplicate cDNA samples were amplified with the SensiFASAT SYBR No-ROX Kit (Bioline) in a Corbet Rotor-Gene 6000 QPCR machine (Qiagen) according to the manufacturer's protocols.
For both cells and adults experiments were performed at least three times independently, giving similar results. All transcript expression values were normalized to actin and were quantified relative to a control using the ΔΔCt method. Following primers were used for QPCR amplification, Upd3 and Soc36E primers as in [18] (link), [33] (link):
attacinA; F 5′-CTCCTGCTGGAAAACATC-3′;
R: 5′-GCTCGTTTGGATCTGACC-3′;
cecropinA1; F: 5′- CATTGGACAATCGGAAGCTGGGTG-3′;
R: 5′- TAATCATCGTGGTCAACCTCGGGC-3′;
diptericin; F: 5′- CCGCAGTACCCACTCAATCA-3′;
R: 5′- TGTGATCTGCAGGATGGTGT-3′;
drosocin; F: 5′- GTTCACCATCGTTTTCC -3′;
R: 5′- CCACACCCATGGCAAAAAC -3′;
puckered;- F: 5′- CATCATCAACGGCAATGAAC -3′;
R: 5′- TTGGGACTTTGGCAGGTAAC -3′;
Rp49; F 5′- CCAGTCGGATCGATATGCTAA-3′;
R: 5′- GTTCGATCCGTAACCGATGT-3′;

Total RNA was extracted from whole flies (6 females), the rest of the body (8 females), and heads (30 females) using the Total RNA Purification Plus kit (Norgen Biotek), and cDNA was prepared from 0.5 μg total RNA using the Maxima First Strand cDNA synthesis kit (Thermo Scientific). Triplicate cDNA samples were amplified with the SensiFASAT SYBR No-ROX kit (Bioline) in a Corbet Rotor-Gene 6000 qPCR machine (QIAGEN) according to the manufacturer’s protocols. Primer sequences are presented in the Supplemental Experimental Procedures.

Drosomycin expression was determined from five independent biological samples consisting of 5 infected third instar larvae following infection. Samples were compared to banana-fed flies of the corresponding time point. Larvae were collected at the desired time points and washed in 100% ethanol and sterile water. RNA was extracted using Purification Plus Kit (48400, Norgen – Biotek, Canada) and cDNA was prepared from 0.5 µg total RNA using Maxima First Strand cDNA Syntesis Kit (K1672, Thermo Scientific, UK). Triplicate cDNA samples were amplified with the SensiFASAT SYBR No-ROX Kit (BIO-98020, Bioline, UK) in a Corbet Rotor-Gene 6000 QPCR machine (Qiagen, UK) according to the manufacturer’s protocols. Expression values were calculated using the DDCt method and normalized to rp49 expression levels (Schmittgen and Livak 2008 (link)).
Primers Used: rp49(forw): AAGAAGCGCACCAAGCACTTCATC, rp49(rev): TCTGTTGTCGATACCCTTGGGCTT, drosomycin(forw): AGTACTTGTTCGCCCTCTTCGCTG, drosomycin(rev): CCTTGTATCTTCCGGACAGGCAGT.