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Gene & Protein in Disease DNA methylation and gene expression on rats with protein malnutrition

ii. Early-life low-protein (LPE) group. From the 1 day total RNA quantity and purity were analyzed using
st
after conception, the mother rats were given a low- Agilent 2100 Bioanalyzer Instrument and RNA 1000
protein diet containing 6% protein to establish the Nano LabChip Kit (Agilent, CA, USA), and the RIN
intrauterine malnutrition LPE rat model. The mother number >7.0. Poly(A) RNA was purified from total RNA
rats were given a 20% protein diet until after weaning. (5 µg) using poly-T oligo-attached magnetic beads using
iii. Fetal low-protein (LPF) group. From the 1 day after two rounds of purification. Following purification, the
st
conception, the mother rats were given a low-protein mRNA was fragmented into small pieces using divalent
diet containing 6% protein to establish the intrauterine cations under elevated temperature. Then, the cleaved
malnutrition LPF rat model until the birth of offspring. RNA fragments were reverse transcribed to create the
After that, the mother and young mice were given a final cDNA library in accordance with the protocol for
20% protein content . the mRNASeq sample preparation kit (Illumina, San
[23]
Diego, USA). The average insert size for the paired-end
In the process of animal experiments, the welfare and
ethical guidelines for experimental animals issued by libraries was 300 bp (±50 bp). Then, we performed the
the Ethics Committee of Animal Experiments of Henan paired-end sequencing on an IlluminaHiseq4000 at the
University were strictly implemented, and the pain of LC Sciences, USA, following the vendor’s recommended
experimental animals was minimized throughout the protocol.
experiment. 2.6. Sequence and primary analysis

2.3. Animal observation and collection of specimens Illumina Hiseq4000 was used for sequencing, and the read
length of sequencing was double-ended 2 × 150 bp. This
During the experiment, litter size, IUGR occurrence, and
perinatal mortality were recorded in each litter, and the yielded gigabases (Gb) of sequence. Before assembly, the
litter birth status of pregnant rats was observed at 8 am, low-quality reads (1, reads containing sequencing adaptors;
2, reads containing sequencing primer; nucleotide with q
12 pm, 4 pm, and 8 pm every day when the rats were close quality score lower than 20) were removed.
to childbirth. Newborn rats born during the daytime were
weighed within 4 h. For nocturnal births, the newborn 2.7. Study on differential expression of genes
rats were weighed no later than 8 a.m. the next day. At the
experimental time point, the weight of the young mice was The study of gene-level expression differences was carried
out based on the data analysis procedure described above.
weighed at a fixed time point (8–9 am).
Ic-bio used Hisat software to compare the sequencing
The rats in the three groups were anesthetized by data to the reference genome and used the alignments to
intraperitoneal injection of 0.3 mL/100 g 10% chloral assemble the transcripts. StringTie, developed at Johns
hydrate at the 48 week, and then, 8 mL blood was collected Hopkins University in association with the University of
th
from the abdominal aorta using a disposable negative Texas Southwestern Medical Center, assembles transcripts
pressure vacuums tube (K2 EDTA anticoagulant tube). and predicts expression levels. It applies network flow
The animals were then sacrificed. RNA was extracted by algorithms and the optional de novo assembly to assemble
TRIzol method and stored at −80°C for future use. After complex datasets into transcripts. Compared to programs
all the animals were sampled, the samples were stored on such as Cufflinks, StringTie achieves more complete
dry ice and transported to Hangzhou Lianchuan Biological and accurate gene reconstruction and better prediction
Company for sample analysis. of expression levels when analyzing simulated and real
datasets. Then, edgeR was used for differential expression
2.4. Sample acquisition and preservation analysis, and R language was used for graphical display
Rats in the three groups were reared until the 48 week, and of differential expression results, including differential
th
8–10 mL blood samples were collected through abdominal expression gene heat map, scatter plot, volcano map, and
aorta puncture before the animals were sacrificed. DNA principal component analysis map.
was extracted by TRIzol method. Blood specimens from
four male and female samples were randomly selected from 2.8. Clustering analysis of differential gene
each group, with 16 samples in total. The blood specimens expression levels
were stored at −80°C. We used log10(FPKM+1) for gene expression
demonstration. At the same time, the gene expression of
2.5. mRNA library construction and sequencing
differentially expressed FPKM (fragments per kilobase of
Total RNA was extracted using TRIzol reagent (Invitrogen, exon per million mapped fragments) can be displayed by
CA, USA) following the manufacturer’s procedure. The Z-value method , where the abscissa is the sample, the
[24]

Volume 1 Issue 2 (2022) 3 https://doi.org/10.36922/gpd.v1i2.169