Quantitative PCR (qPCR) was performed to characterize the relative Wolbachia infection level in the S2 cell lines and flies. The protocol was similar to prior qPCR amplification using the single-copy wsp and su(fk)C genes of bacterial and host origin, respectively [65 (link)]. S2 cells were quantified using a hemocytometer to obtain 106 cells. The S2 cells or DSR females were homogenized in 100 μl STE with 0.4 mg/ml proteinase K to extract DNA as previously described [66 (link)].
For qRT-PCR, RNA extractions were performed on groups of 10 ovaries or 10 testes dissected from one-day post eclosion infected and uninfected Drosophila adults using the RNeasy Mini Kit (Qiagen). DNA contamination was removed with RNase-Free DNase Set (Qiagen). RNA quality and quantity was checked with NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Inc.). Synthesis of cDNA was performed with Superscript II Reverse Transcriptase (Invitrogen) using specific primer for Ance (AnceQ F 5'-CGGTCACGTTCGATTGGTTG-3' and AnceQ R 5'-CTTCGGTTTCCACGTTGGTTC-3') and Actin gene (ActinQ F 5'-GCTGACCGTATGCAAAAGG-3' and ActinQ R 5'-GCTTGGAGATCCACATCTG-3'). Primers were designed based upon D. simulans genbank sequences for Ance and Actin (genbank accession number: NM_057696 and NM_079486, respectively]. qRT-PCR was performed separately with the AnceQ F/R and ActinQ F/R primer pairs using a Miniopticon system (BioRad) with a Platinum SYBR Green qPCR superMix (Invitrogen). qRT-PCR reactions were conducted using a 2 minute step at 50°C, 2 minute step at 95°C and 40 cycles of 15 seconds at 95°C and 30 seconds at 56°C. A fluorescence measurement was made at the end of each cycle. A melting curve analysis was performed at the end of the amplification program to examine for primer-dimers or nonspecific amplification. Assays were performed on two (D. simulans and D. melanogaster wild type) or three (D. melanogaster Ance mutants) independent experiment replicates for each sex and infection type. As an examination for variability, duplicate qRT-PCR reactions were performed for each set of ovaries or testes with both the Ance and Actin primers. Relative expression of Ance gene was calibrated against Actin using the ΔΔCT calculation method [67 (link)] with:
For comparisons of males and females, the above was modified as follows:
For qRT-PCR, RNA extractions were performed on groups of 10 ovaries or 10 testes dissected from one-day post eclosion infected and uninfected Drosophila adults using the RNeasy Mini Kit (Qiagen). DNA contamination was removed with RNase-Free DNase Set (Qiagen). RNA quality and quantity was checked with NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Inc.). Synthesis of cDNA was performed with Superscript II Reverse Transcriptase (Invitrogen) using specific primer for Ance (AnceQ F 5'-CGGTCACGTTCGATTGGTTG-3' and AnceQ R 5'-CTTCGGTTTCCACGTTGGTTC-3') and Actin gene (ActinQ F 5'-GCTGACCGTATGCAAAAGG-3' and ActinQ R 5'-GCTTGGAGATCCACATCTG-3'). Primers were designed based upon D. simulans genbank sequences for Ance and Actin (genbank accession number: NM_057696 and NM_079486, respectively]. qRT-PCR was performed separately with the AnceQ F/R and ActinQ F/R primer pairs using a Miniopticon system (BioRad) with a Platinum SYBR Green qPCR superMix (Invitrogen). qRT-PCR reactions were conducted using a 2 minute step at 50°C, 2 minute step at 95°C and 40 cycles of 15 seconds at 95°C and 30 seconds at 56°C. A fluorescence measurement was made at the end of each cycle. A melting curve analysis was performed at the end of the amplification program to examine for primer-dimers or nonspecific amplification. Assays were performed on two (D. simulans and D. melanogaster wild type) or three (D. melanogaster Ance mutants) independent experiment replicates for each sex and infection type. As an examination for variability, duplicate qRT-PCR reactions were performed for each set of ovaries or testes with both the Ance and Actin primers. Relative expression of Ance gene was calibrated against Actin using the ΔΔCT calculation method [67 (link)] with:
For comparisons of males and females, the above was modified as follows:
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