Rnalater stabilization solution

To prevent RNA degradation, bacterial cells cultivated in LB broth or animal cells infected with Salmonella were treated with RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) or RNAlater stabilization solution (Ambion, Austin, TX, USA) according to the manufacturer's instructions. Bacterial total RNAs were isolated using RNeasy mini kit (Qiagen) and residual chromosomal DNAs were removed using TURBO DNA-free kit (Ambion). RNA was reverse-transcribed into cDNA using RNA to cDNA EcoDry Premix with random hexamers (Clontech Laboratory, CA, USA), and cDNA corresponding to 10 ng of input RNA was used as a template in each qPCR. Primers were designed using Primer Express Software ver. 3.0 (Applied Biosystems, MA, USA) and their sequences are listed in Table S2. qPCR was conducted using a StepOnePlus real-time PCR instrument (Applied Biosystems) with SYBR green reagent (Power SYBR Green PCR Master Mix, Applied Biosystems) to detect the amplified PCR product. Relative expression levels of each gene were normalized to those of gyrB (28 (link)) and are presented as the average from three tests using independently extracted RNA samples.

Tumor samples (n = 26) were obtained from the patients undergoing gastrectomy surgery at Al-Zahra Hospital, Isfahan, Iran, and Kashani Hospital, Shahre Kord, Iran. In addition, endoscopy samples (n = 11) were taken from healthy volunteers and used as control samples. Immediately after obtaining tissue samples, they were submerged in a 2 ml RNase-free containing RNAlater™ Stabilization Solution (Ambion, Austin, Texas) and transferred to a laboratory on ice for subsequent analyses. Before specimen collection, the stomach biopsies were taken to detect H. pylori infection using Rapid Urease Detection test (HelicotecUT Plus, Taiwan) in both patient and control groups. The normal/tumor status of the samples was verified by four expert pathologists. Ethically, the samples were collected in accordance with the guidelines issued by the Ethics Committee of Al-Zahra Hospital, regarding 1964 Helsinki declaration. Moreover, informed consent was gained from all studied individuals. The characteristics of the samples including age, sex and H. pylori infection are listed in Table 1.
Table 1

Patient sample characteristics

Variable		Control (n = 11)	Case (n = 26)	p
Age (SD)		49.73 (21.45)	61.13 (13.54)	0.055 *
Sex	Woman	4	6	0.406 **
	Man	7	20
H. pyloriinfection	Negative	5	12	0.969 **
	Positive	6	14

SD Standard deviation, H. pylori Helicobacter pylori

* Independent t-test

** Chi-square test

Mice (n = 32) were intraperitoneally injected with 7.4 mg/kg body weight of AOM (Sigma-Aldrich, St Louis, MO, USA) dissolved in phosphate-buffered saline (PBS). Five days after AOM administration, 1% DSS (MP Biomedicals, Santa Ana, CA, USA) was added to the animals’ drinking water for 1 week, and then water without DSS was provided for 2 weeks (first cycle). Each cycle consisted of 1 week of DSS treatment and a 2-week of recovery period. The control group (n = 27) followed the same protocol excluding the AOM injection on day 0. Body weight was measured once every week. Mouse colon tissues were harvested before and after each DSS treatment throughout the study duration (Fig. 1A). Upon opening the colon, the entire luminal surface was observed and tumors (greater than 1 mm in diameter) were enumerated. Approximately 1 cm of distal colon was bisected longitudinally; half was stored in RNAlater® stabilization solution (Ambion, Austin, TX, USA) for RNA analysis and the other half was fixed in 4% paraformaldehyde in neutral buffer solution for paraffin embedding.

Gene expression levels were analyzed by semi-quantitative real-time PCR. Mouse heart was quickly removed after sacrifice; ventricular apex tissue was sampled, stored in RNAlater Stabilization Solution (AM7024 Ambion, USA) at −80 °C until use. Total RNA was extracted with RNAiso PLUS (9108 Takara Bio, Japan), followed by the further purification with RNeasy mini-kit (74104 Qiagen, USA) according to the manufacturer’s instruction. Two μg of total RNA was then used to synthesize first strand cDNA with a SuperScript VILO cDNA Synthesis Kit (11754050 Invitrogen, USA). mRNA levels were quantified by real-time PCR with SYBR green dye (QPS-201 Thunderbird Sybr qPCR Mix Toyobo, Japan) with the specific primer sets^{13 (link),38 (link),39 (link)} shown in Table 2, and normalized to ribosomal protein L4 (RPL4) mRNA^{40 (link)}.

In order to isolate high-quality RNA, the rectal biopsy was taken by colonoscopy and immediately submerged in 0.5 ml of RNA later stabilization solution (Ambion, Austin, TX, USA) for storage and stored at -80°C until used. Then, total RNA was isolated using High Pure RNA Tissue (Roche Diagnostics, Mannheim, Germany) [6 (link), 7 (link)].
For q-PCR assay quality control, determination of linearity and reproducibility was evaluated (VC < 10%). The mRNA relative quantification of target genes was conducted using the Light Cycler software 4.1, according to the 2ΔΔCt method. Table 1 shows the details of the primer's designs and number of UPL (Universal Probe Library; Roche Diagnostics, Mannheim, Germany) used for the RT-PCR assay.

For the initial identification of viroids and viroid-like RNAs from apple, leaves were collected from an apple (Malus pumila Mill. cv. Fuji) plant, the fruits of which showed typical symptoms of apple scar skin viroid disease, in Shandong province China, in July 2012. The grapevine (Vitis vinifera L.) leaf samples used for determination of GLVd were from Tulufan in Xinjiang China. This grapevine plant (cv. Thompson seedless) for sample collections is more than 100 years old. Young leaves of both apple and grapevine were immediately put into RNAlater stabilization solution (Ambion, USA) after collection and sent to a laboratory for deep sequencing analysis. Moreover, approximately 10 g of apple and grapevine leaves were packaged with ice, kept fresh at low temperature, and sent to a laboratory for RNA analysis using PAGE and northern-blot hybridization.
To survey the occurrence of viroid-like apple RNA in China, 182 leaf samples of variant apple cultivars from five provinces were collected from 2012 to 2014 and kept at −80°C.