Precellys lysing kit ck14

The tissue was transferred from the cryovials into beads-filled tubes (Precellys^® Lysing Kit CK 14, Bertin, France). The CN of the animals of one age group were pooled in one bead tube (n = 4 animals and 8 CN per age group) without a medium. Pooling was necessary to generate enough mRNA for further analysis. Weighing of pooled CN was performed (Sartorius^® Handy M160, Goettingen, Germany). The pooled CN weighed less than 20 mg, regardless of age. Therefore, following the instructions of the RNeasy Mini Kit (Qiagen^®, Venlo, The Netherlands), 350 µL of RLT buffer (Qiagen^®, Venlo, The Netherlands) was added per tube. These were homogenized in two homogenizer steps (Precellys 24 DUAL^®, Bertin, France) at 6000 rpm for 30 s each. A total of 350 µL of ethanol 70% (Thermo Fisher Scientific^®, Waltham, MA, USA) was added to the resulting emulsion. Further steps were performed according to the instructions of the RNeasy Mini Kit (Qiagen^®, Venlo, The Netherlands).
Subsequently, the extracted RNA was quantified using a spectrophotometer (NanoDrop One/One^c, Thermo Fisher Scientific^®, Waltham, MA, USA), and its purity (A260/A280) was determined. At postnatal day 6 (p6), animals had approximately 350 ng/mL RNA. p12 and p24 animals had about 750 ng/mL. Only RNA with an A260/A280 ratio of 2.0 ± 0.1 was used to synthesize complementary DNA (cDNA).

The hippocampus was homogenized using Precellys lysing Kit CK14 (Bertin Instruments) and Precellys homogenizer (Bertin Instruments). RNA from hippocampus was isolated using Direct-zol RNA MiniPrep (Zymo Research), according to the manufacturer’s protocol. SuperScrip III Reverse Transcriptase (Invitrogen, USA) and random hexamers were used to synthesize cDNA according to the manufacturer’s protocol. Taqman Gene Expression Mastermix (Thermo Fisher Scientific) and following TaqMan Gene Expression Assays were used for real-time quantitative PCR: Ip10 (Rn01413889_g1), Ki67 (Rn01451446_m1), Tlr2 (Rn02133647_s1), Tlr4 (Rn00569848_m1), Chop (Rn00492098_g1), Grp78 (Rn00565250_m1) and Hmox1 (Rn00561387_m1). Hprt1 (Rn01527840_m1) was used as an internal control and 2^−∆Ct method was used for relative quantification.

The lower lung lobe and left ventricle of the heart were homogenized using a Precellys lysing Kit CK14 (Bertin Instruments, Montigny-le-Bretonneux, France) and a Precellys homogenizer (Bertin Instruments). The total RNA from the tissue lysates was isolated using Direct-zol RNA MiniPrep (Zymo Research, Irvine, CA, USA) and from the rat primary cortical neurons using the Qiagen RNeasy Mini Kit, according to the manufacturers’ protocol. The RNA was reverse-transcribed using random hexamers and SuperScript™ III Reverse Transcriptase (Invitrogen, Waltham, MA, USA).
The qPCR was performed on a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystem, Waltham, MA, USA) using the Taqman Gene Expression Mastermix (Thermo Fisher Scientific, Waltham, MA, USA) and the following TaqMan Gene Expression Assays: Ace (Rn00561094_m1), Ace2 (Rn01416293_m1), Agtr1a (Rn02758772_s1), Agtr1b (Rn02132799_s1), Agtr2 (Rn00560677_s1), Bdkrb1 (Rn02064589_s1), Bdkrb2 (Rn01430057_m1), Mas1 (Rn00562673_s1) and Wfs1 (Rn00582735_m1). In all the gene expression experiments, the amount of the target gene was normalized to Hprt1 (Hypoxanthine-guanine phosphoribosyltransferase; Rn01527840_m1) or Tbp (TATA box binding protein, Rn01455648_m1) as an endogenous reference control by means of the 2^−ΔCt method [50 (link)].