Nanodrop spectrophotometer nanodrop nd 1000

Blood sample was diluted with equal amount of sterile phosphate-buffered saline (PBS) solution (GE Healthcare Life Sciences). One milliliter of Ficoll-Paque (GE Healthcare Life Sciences) was inserted into a 2 ml Eppendorf centrifuge tube. Diluted blood sample was layered carefully onto the Ficoll-Paque. Then sample was centrifuged at 400 gav for 30 min at 18 °C. After removing an upper layer, a lymphocyte layer was transferred into a new 2 ml Eppendorf tube, filled with PBS solution, mixed and afresh centrifuged at 100 gav for 10 min at 18 °C. Then the supernatant was discarded carefully not to disrupt the pellet of the peripheral blood mononuclear cells (PBMC). Subsequently, PBMCs were washed again with PBS solution (centrifugation repeated under the same conditions). Afterwards 800 µl of lysis solution and 50 µl of sodium acetate (Mouse RiboPure™-Blood RNA Isolation Kit; Invitrogen; Thermo Fisher Scientific) was added, the cells were suspended by manual pipetting, then the samples were placed immediately at −80 °C. Total RNA was isolated with the use of Mouse RiboPure™-Blood RNA Isolation Kit (Invitrogen; Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA concentration was measured with the use of NanoDrop Spectrophotometer (NanoDrop ND-1000; Thermoscientific, Waltham), and RNA quality was determined by TapeStation 4200 (Agilent, Santa Clara).

Microbial genomic DNA was extracted from 180 mg of colonic digesta samples using the QIAamp DNA Stool Kit in accordance with the manufacturer’s instructions (Qiagen, West Sussex, UK). The DNA purity/integrity was determined via 1% agarose gel electrophoresis (Bio-rad Laboratories Ltd., Watford, Hertfordshire, UK), ethidium bromide staining (Sigma-Aldrich) and UV visualisation (LumiBIS, DNR bioimaging systems, Israel). The quantity of DNA was assessed using a Nanodrop spectrophotometer (NanoDrop ND-1000; Thermo Scientific, Wilmington, DE, USA). The samples were stored at −20 °C.

After obtaining written informed consent from the parents, blood samples (0.3 mL) were drawn from all the study participants on the 5th and 28th day of life (DOL) for the assessment of whole genome expression in peripheral blood leukocytes. Subsequently, Ficoll isopaque gradient centrifugation (30 min, 2100 rpm, RT), two times wash in 1x PBS (12 min, 1600 rpm, 40°C), and finally RiboPure Blood Kit (Ambion, Life Technologies, Carlsbad, USA) were used for total RNA extraction. RNA concentration was measured with the use of NanoDrop Spectrophotometer (NanoDrop ND-1000; Thermoscientific, Waltham, USA), and RNA quality was determined by 2100 Bioanalyzer (Agilent, Santa Clara, USA).
100 ng of total RNA was used for the single microarray experiment. GeneChip Human Gene 1.0 ST Arrays (Affymetrix, Santa Clara, USA) were used. Whole transcript microarray experiment was performed. Details of microarray experiment were published previously [10 (link)].