Rneasy protect mini kit

DRG were dissected as previously described (refer to DRG extraction section) and were collected in an Eppendorf tube containing 200 μL/tube of RNA-Later solution. Tubes were left at −20 °C overnight and the day after tissue was crushed with a pestle and lysed by adding 200 μL/tube of freshly prepared lysis buffer (RNeasy Protect Mini Kit, Qiagen) and mercaptoethanol. RNA was extracted following standard procedure using the RNeasy Protect Mini protocol (Qiagen) including DNase I digestion step. Total RNA extracted was converted to first-strand cDNA using the SuperScript III First-Strand Synthesis System kit (Thermo Fisher Scientific). For HEK-293 cell experiment, cells were lysed by adding 600 μL/well of freshly prepared lysis buffer (RNeasy Protect Mini Kit, Qiagen) and mercaptoethanol. Cell lysates were transferred to Eppendorf tubes and RNA was extracted following standard procedure using the RNeasy Protect Mini protocol (Qiagen) including DNase I digestion step. Total RNA extracted was converted to first-strand cDNA using the SuperScript® III First-Strand Synthesis System kit (Thermo Fisher Scientific).

Twenty male and females were removed, respectively, at intervals during feeding to recover material representing unfed (day 0 weighing < 5 mg), early feeding (day 2 weighing 6–15 mg for females and < 5 mg for males), mid feeding (day 4 weighing 16–23 mg for females and ~ 5 mg for males) and late feeding (day 6 weighing > 24 mg for females and ~ 7 mg for males) ticks. Salivary glands were dissected out within two hours of tick removal, cleaned from other internal tissues and placed into at least ten times its volume RNAlater (Qiagen, AMBION, Inc., Austin, Texas), and left overnight at 4 °C before storing at -70 °C. Each day’s samples were pooled by gender to render one male (40 glands) and one female (40 glands) sample per time point. Salivary glands were suspended in 600 µl RLT buffer (RNeasy Protect Mini Kit) per 20 mg tissue and total RNA extracted using the RNeasy Protect Mini Kit (QIAGEN Group). Residual genomic DNA was removed with DNase I digestion (QIAGEN Group). Total RNA quantification and assessment of the integrity of RNA was done using the Qubit fluorometer 2.0 (Life Technologies, Carlsbad, CA) and the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).

Bone marrow samples were stored at -80 °C after homogenization in RTL buffer (QIAGEN, 79216). Total RNA was isolated with the QIAGEN RNeasy Protect Mini Kit (QIAGEN, 74104). RNA samples were treated with DNase (1 U/µg, Sigma, AMPD1-1KT) for possible DNA contaminations during isolation. One microgram of total RNA was used for cDNA synthesis by random hexamers and MMLV reverse transcriptase (MBI Fermentas, EPO351) according to the recommendations of the manufacturer.