6- to 8-week-old female CB6F1 mice were purchased from Charles River. Animals received a single injection in their right hind leg quadricep muscle and were then culled at specific intervals after the vaccination, either 4, 8, 24, 48, 72 or 168 Hrs. A control group that did not receive a vaccination was culled and tissues harvested. A further control group was employed that received a single saline injection in the right hind leg quadriceps muscle and were culled at the same intervals as the animals that received an active formulation. When each animal was culled the injected muscle site and the iliac lymph nodes that drain the hind leg quadriceps muscles were harvested and flash frozen in liquid nitrogen. Peripheral blood, sampled from the mouse tail vein immediately before humane euthanasia was collected (100 µL) in RNAprotect animal blood tubes (Qiagen, UK). Groups of mice (n = 5 per group per timepoint) received 1/10th of the human dose in 50 µL of one of the following licensed vaccines: Pentavac SD (Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine); Agrippal (Trivalent Flu Subunits – H3N2, H1N1 and Influenza B); Fluad (Trivalent Flu Subunits – H3N2, H1N1 and Influenza B + MF59® (oil-in-water emulsion)); Engerix B (Recombinant Hepatitis B) and either Poly I:C (Sigma, UK – P0913: 50 µL of a 1 mg/mL solution), LPS (Invivogen, UK – LPS-EB Ultrapure: 50 µL of a 0.5 mg/mL solution), IFA (Seppic, France – Montanide ISA 51 VG: 50 µL of a 1:1 mixture of IFA and Saline) or Saline alone (Sigma, UK – 50 µL).
Extracted molecule
total RNA
Extraction protocol
Tissue Samples - Small pieces of tissue (3mm x 3mm x 3mm) were harvested and flash frozen in liquid nitrogen. Total RNA isolation including the miRNA species was performed using the miRNeasy mini kit (Qiagen, UK), as described in the standard protocol for purification of microRNA and total RNA from tissues and cells. Briefly, 700 µL QIAzol lysis reagent was added to the tissue sample, and disrupted and homogenized using a tissueruptor probe. Homogenate was incubated at room temperature (15–25°C) for 5 min before addition of 140 µL chloroform and vigorous shaking for 15 s. After incubation at room temperature for 2–3 min the homogenate was centrifuged for 15 min at 12,000 x g at 4°C. The upper aqueous phase was then transferred to a new collection tube (350 µL). 1.5 volumes (525 µL) of 100% ethanol was added and mixed thoroughly by pipetting then all sample was transferred into an RNeasy® Mini column, the sample being pulled through the column by vacuum manifold. The sample on the column was washed using 700 µL Buffer RWT, followed by 2 washes using 500 µL Buffer RPE. The RNeasy Mini column was then placed into a new 2 mL collection tube and centrifuged at full speed for 1 min to completely dry the membrane. The RNeasy Mini column was then transferred to a new 1.5 mL collection tube and 30 µL RNase-free water was directly pipetted onto the column membrane, then centrifuged for 1 min at ≥8000 x g to elute. Purified RNA was stored at -80°C until required for microarray hybridisation. Blood Samples - 100 µL of peripheral blood harvested from the mouse tail was collected directly into an RNAprotect Animal Blood Tube and the tubes incubated at room temperature (15–25°C) for 2 hours. The blood tubes were then flash frozen in liquid nitrogen for storage. RNA isolation was performed as described in the kit protocol for purification of Total RNA, including miRNA, from RNAprotect stabilized animal blood tubes (100 µL). Briefly, completely thawed tubes were first centrifuged for 3 min at 5000 x g, then the supernatant removed by careful pipetting. The pellet was resuspended in 1 mL of RNase-free water and transferred to a clean RNase-free 2 mL tube. The pellet was completely resuspended in the water, centrifuged for 3 min at 5000 x g and the supernatant carefully removed by pipetting. The pellet was then dissolved in 240 µL Buffer RSB before addition of 200 µL Buffer RBT and 20 µL proteinase K enzyme solution. The tube was vortexed for 5 s then incubated at 55°C for 10 min in a shaking incubator at 1200 rpm. After incubation the sample was added into a Qiashredder spin column and centrifuged for 3 min at 10,000 x g. The supernatant was then transferred to a clean tube and 690 µL 100% ethanol added, mixed thoroughly by pipetting then all sample was transferred into an RNeasy Mini column, the sample being pulled through the column by vacuum manifold. The sample on the column was washed using 350 µL Buffer RWT, followed by addition of 80 µL DNase I solution (in Buffer RDD) and incubation at room temperature for 15 min. After DNase 1 treatment the column was washed with a further 350 µL of RWT followed by 2 washes using 500 µL Buffer RPE and a final wash with 500 µL 80% RNase-free ethanol. The RNeasy Mini column was then placed into a new 2 mL collection tube and centrifuged at full speed for 1 min to completely dry the membrane. The column was then transferred to a new 1.5 mL collection tube and 30 µL RNase-free REB buffer was directly pipetted onto the column membrane, then centrifuged for 1 min at ≥8000 x g to elute. Purified RNA was stored at -80°C until required for microarray hybridisation.
Label
not provided
Label protocol
Gene expression data were generated from high-quality RNA samples on an Agilent microarray platform (Agilent Technologies). RNA was labeled with a Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions.
Hybridization protocol
Quantity and labeling efficiency were verified before hybridization to whole-genome 8×60 k mouse expression arrays (Agilent design ID 028005).
Scan protocol
Scanned at 5 μm using an Agilent scanner.
Description
m.muscle.TriFlu.08.4 Gene expression in muscle 8hrs after injection with Agrippal.
Data processing
Image analysis and data extraction were performed with Agilent's Feature Extraction software (version 11.5) to generate the raw expression data. Microarray data were pre-processed and normalised using R package limma. The raw data were first background corrected using the normexp method. Background corrected signals were quantile normalised between arrays.
