Isolation of RNA from the murine colonic tissue and qRT-PCR for inflammatory cytokines

Abstract

E-cigarette (E-cig) inhalation affects health status by modulating inflammation profiles in several organs including the brain, lung, heart, and colon. The effect of flavored 4^th generation pod-based E-cigs (JUUL) on murine gut inflammation is affected by both flavor and exposure period. Exposure of mice to JUUL Mango and JUUL Mint for 1 month upregulated inflammatory cytokines, particularly TNF-α, IL-6, and Cxcl-1 (IL-8). JUUL Mango effects were more prominent than those incurred by JUUL Mint after 1 month of exposure. However, JUUL Mango reduced the expression of colonic inflammatory cytokines after 3 months of exposure.  In this protocol, we detail the process of RNA isolation from mouse colon and the use of extracted RNA in profiling the inflammatory milieu. Efficient RNA extraction from the murine colon is the most important step in the evaluation of inflammatory transcripts in the colon.  

For complete details of this paper and additional methods, please refer to Moshensky A et al. (Moshensky et al., 2022)

Keywords: e-cigarette, flavored JUUL, mouse colon, RNA, and inflammation.

Background

E-cigarettes (E-cigs) were first introduced to the international market in the mid-2000s as an alternative to conventional tobacco smoking (O'Loughlin et al., 2016).  E-cigs produce an aerosol (commonly called vapor) upon heating and aerosolization of vehicle solvents propylene glycol (PG) and vegetable glycerin (VG). In addition, a large variety of flavors are added to E-cigs to add appeal to people of all kind, including women, children and minorities (Zhu et al., 2014).

JUUL is one of the most popular pod-based E-cig brands. They sale pods containing e-liquids with different flavors such as Mint and Mango (Huang et al., 2019). The effect of chronic inhalation of the aerosols produced from these devices on heath is not yet understood. 

Moshensky A et al. used in vivo mouse exposures of daily JUUL aerosol inhalation with different flavors (Mango and Mint) for 1 and 3 months to evaluate the effects of JUUL aerosol inhalation on the function and inflammation of different organs (Moshensky et al., 2022). The authors found that the JUUL aerosol inhalation induced inflammation in the brain, gut, and heart (Moshensky et al., 2022). A recent study using the stem cell based approach of 3D gut organoids derived from healthy individuals revealed that E-cig induced inflammation in the gut epithelium and damaged epithelial tight junctions (Sharma et al., 2021).

Extraction of RNA from the murine colon is the most important step in the relative quantification of the colon transcripts. Several factors affect the quality of the extracted RNA used in qRT-PCR such as the method used in the extraction, RNA purity and concentration, and presence of other impurities including guanidinium isothiocyanate, phenolic compounds, ethanol, tissue DNA, and protein. The first two impurities are used during the RNA extraction to aid in the isolation and protection of RNA, and ethanol is added to the washing buffers during the extraction procedure. However, the excess presence of these substances could affect the downstream processing such as cDNA synthesis and PCR (Toni et al., 2018). In addition, tissue DNA and protein are other contaminants to the extracted RNA. In this protocol, we describe the detailed steps of efficient isolation of RNA from the colon of mice exposed to E-cigs with minimized levels of contamination that affect downstream processing. Also, we describe the process of quantitative measurement of the transcripts of inflammatory cytokines in the extracted RNA.  

Materials and Reagents

a) Materials 

b) Regents

Equipment and software
a. Equipment

b.  Software

Protocol 

A. Isolation of colon from the exposed mice (Figure 1)

1. Female C57BL/6 mice (6-8 weeks) were exposed to E-cig aerosols produced from JUUL Mango, JUUL Mint or Air for 1 and 3 months using the InExpose system (Scireq).
   Note: Detailed protocol about mouse exposures and composition of JUUL pods are present in the original article (Moshensky et al., 2022).
2. At the end of exposure, mice were euthanized by accessing the peritoneal space and terminal bleeding via the inferior vena cava under deep anesthesia, followed by opening of the thorax. 
3. Using scissor and forceps, cut the whole colon of a mouse from the anus to the cecum (Figure 1).
4. Put the colon in a petri dish containing PBS to rinse away blood. Remove any attached adipose tissue from the colon.
5. Use a cotton swab to compress along the length of the colon to empty the colon of remaining stool.
6. Cut the colon into small pieces and transfer part of the colon into 1.5 ml Eppendorf containing 400-500ul of RNAlater. 
   Note a) Saving of the whole colon in RNAlater solution is an option if no other procedure/ technique is required such as processing of the mouse colon into fixed formalin paraffin embedment (FFPE) blocks. Therefore, step #6 can be optional. 
   b) In case histology is required for the colon, cut the mouse colon into 3 parts: proximal colon, mid colon, and distal colon. The distal colon is used to assess the transcripts of inflammatory cytokines. Mid colon and proximal colon are used for histology and other purposes.
7. Store the colon samples at -80ᵒC.
   Note: If the RNA extraction will be done on the same day of euthanasia, step #7 is not required. Saving the tissue in RNAlater is important when the downstream processing will be performed later. 

B. Isolation of RNA from the colon tissues (Figure 2).

1. Thaw the colon samples on ice, then use scissors and forceps to cut the colon tissues into small pieces.
   Note: Be sure that no precipitated salts are around the tissue. Wash the colon with phosphate buffered saline (PBS) to remove any precipitated salts.
2. Weigh the colon piece using a scale/balance and be sure that the weight is ≤ 20 mg/piece.
   Note: a) It is very important to weigh the mouse colon, ideally tissues mass between 5-20 mg give good RNA yield. Using larger tissue pieces gives a poor RNA yield due to the column blockage during the RNA extraction process.
   b) It is recommended to mince/cut the colon into small pieces after weighing to speed up the homogenization step.
3. Transfer the colon piece into 1.5 ml Eppendorf containing 600 ul of TRI Reagent solution.
4. Homogenize the colon piece in TRIL using homogenizer 150 for 1-2 minutes till complete disruption of tissue.
   Note:   a) It is important to clean and disinfect the homogenizer using 70% ethanol and water between sample to sample to avoid the cross contamination  
   b)  Make sure that the weighted piece is completely disintegrated, and no visible tissue is remaining.
   c) In case of tough tissue, you can increase the volume of TRI regent to 800 ul and/or increase the homogenization time.
5. Centrifuge the tissue suspension at maximum speed 13,000 x g for 2 min. 
6. After the centrifugation step, you can see a precipitated debris at the bottom of Eppendorf and a clear supernatant solution above it (Figure 2). Collect the supernatant into a new 1.5 ml Eppendorf and discard the tube containing the precipitated debris 
7. Mix the supernatant from step # 6 with an equal volume (about 600 ul) ethanol (95-100%). 
8. Transfer the previous mixture into a Zymo-Spin™ IICR Column in a Collection Tube. 
   Note: The maximum capacity of Zymo-Spin™ IICR is 700 ul, and the volume of each sample is about 1200 ul (600ul TRI/ tissue mix+ 600 ul ethanol). Therefore step # 2 is repeated twice on the same column
9. Centrifuge the mixture from step #8 at 13,000 xg for 1 min. Then discard the flow-through waste into 15 ml or 50 ml falcon tubes and discard the collection tube. 
   Note: At this step, the flow-through waste can be used to purify the protein from the mouse colon. 
10.  Transfer the Zymo-Spin™ IICR Column into a new collection tube and repeat steps # 8 and 9.
11.  Add RNA Wash Buffer (400 µl/ sample) to the column and centrifuge at 13,000 x g for 30 seconds- 1 minutes. Discard the flow-through and transfer the column into a new collection tube.
12.  Add 80 µl of DNase I/ digestion buffer solution per column (recipe 1). Incubate the mixture with the column at room temperature for 15 minutes
    Note 
    a) DNase I is provided as lyophilized powder. Reconstitute it using with DNase/RNase-Free Water according to the manufacturer’s instructions.  The volume of added water depends on the unit in DNase I powder. For example, add 275 µl water for 1500 U of DNase I powder and 55 µl water for 250 U of DNase I powder. Mix well and then save the aliquots in -20ᵒC till use.
    b) This step is highly recommended to get rid of DNA present in the sample.  
13. Add 400 µl Direct-zol™ RNA PreWash buffer to the column and centrifuge at 13,000 x g for 1 minute. Discard the flow-through and repeat this step one more time 
    Note: RNA PreWash buffer is provided as a concentrate buffer. You should dilute it with ethanol according to the manufacturer’s instructions.
14.  Add 700 µl RNA Wash Buffer the column and centrifuge at 13,000 x g for 1 minute to remove the wash buffer
    Note: RNA Wash buffer is provided as a concentrate buffer. You should dilute it with ethanol according to the manufacturer’s instructions.
15. Centrifuge the empty column present in a new collection tube at 13,000 x g for 5 minute to ensure complete removal of the ethanol that present in RNA PreWash buffer and RNA Wash Buffer.
    Note: It is important to get rid of ethanol since any residual amount remaining can impact the downstream qPCR steps and/or the yield and purity of extracted RNA.
16. Transfer the column carefully into an RNase-free tube, add 50 µl of DNase/RNase-Free Water directly to the column matrix, incubate at room temperature for 2-5 minutes and then centrifuge at 13,000 xg for 1 minute.
17. Repeat step #16 using the RNA eluted from the column. Transfer the eluted RNA into the same column, incubated at room temperature for 2 minutes and then centrifuge at 13,000 xg for 1 minute. 

C. RT-qPCR for the inflammatory transcript in the mouse colon

   1.1 conversion of RNA into cDNA

1. The extracted RNA (about 500 ng-750 ng) is converted into cDNA using qScript cDNA SuperMix (Quanta bio) according to manufacturer’s instructions.
2. In 0.2 ml thin-walled PCR tube placed on ice, add the following components as shown in (recipe 2). The volume of RNA added depends on the concentration. Total RNA added should be added 500-750 ng. The volume of free water added depends on the volume of RNA added according to recipe 2
   Note: It is preferred when compared samples with each other, to use the same amount of RNA. For examples: to compare the transcripts from mouse colon derived from air-exposed groups and from JUUL Mango pods, use the same amount of RNA to form cDNA either 500 ng for all samples or 750 ng for all samples, or in between concentrations. 
3. Vortex the mixture gently, and then centrifuges for 10 seconds to collect contents.
4. Place the PCR tubes in a MiniAmp Plus Thermal Cycler programmed as in recipe 3.
5. Dilute the cDNA product into 1/5 or 1/10 using nuclease-free water and save the diluted cDNA in – 20 °C till analysis.

1.2 qRT-PCR reaction system

1. The synthesized cDNA is used as a template for qPCR reaction with 2x Bimake^TM SYBR Green Master Mix.
2. Using a MicroAmp™ Optical 96-Well Reaction Plate, we added the PCR reaction mixtures according to recipe 4. 
3. Add the adhesive plastic tape on the plate, put in the machine and run the PCR program (recipe 5)
    

Data analysis

High yield purified RNA extraction is possible from mouse colon tissue. Using the RNAlater solution helps preserve tissues and/or the RNA for a longer period at -80ᵒC (Figure 1). The previous step aids in the extraction of many samples at the same time to minimize the inter-assay procedure. It is important to weigh the colon piece undergoing the RNA extraction process (Figure 2). Since each column has a maximum capacity, including too large of a sample could lead to blockage of the column and therefore negatively impact all the downstream process of RNA extraction. The addition of TRI reagents stops RNase enzymes and neutralizes any infectious agents in the samples; therefore, it helps in improving the quality and stability of extracted RNA. Following the previously described procedure, you can assess the concentration and purity of RNA using a nanodrop spectrophotometer. For the concentration, you may obtain ~100 ug of total RNA from the previous procedure. The purity of extracted RNA can be assessed by the measurement of absorbance (A_260/280 and A_260/230). The acceptable value for pure RNA in terms of A_{260/280 is} 1.8-2.2 or a value >1.8.  A lower value than 1.8 indicates the presence of contaminants that absorb at 280 nm such as protein and phenol. Similarly, 260/230 values >1.8 indicates pure RNA, while values lower than 1.8 indicate contamination with TRI reagent that absorbs at 230 nm.

For the qRT-PCR, we determined the cycle threshold (Ct) value for the target gene, which is then normalized to the housekeeping gene (∆CT). For comparison of the relative transcript expression between the different groups of mice, we used the formula ∆∆CT. For example, to compare the relative IL-6 transcript expression, we determined

 ∆CT for IL-6 in JUUL exposed mice = CT _IL6- CT _{housekeeping gene}).

∆CT for IL-6 in air-exposed mice = CT _IL6- CT _{housekeeping gene})

∆∆CT= ∆CT for IL-6 in JUUL exposed mice- ∆CT for IL-6 in air-exposed mice

Notes

1. This method is suitable for RNA extraction from different mouse organs
   Although the previous protocol focused mainly on the mouse colon, it can be applied to extract RNA from different mouse organs such as the spleen, liver, brain, lung, etc.  We previously used this protocol to assess the inflammatory responses in the liver, spleen, cecum, and intestine of mice infected with Salmonella infection (Sayed et al., 2021). The liver and spleen are thicker than the intestine, therefore the volume of TRI reagent should be increased to 800 ul and the homogenization time should be increased to 3-5 minutes.
2. The quality of RNA and PCR products is important in the interpretation of the results.
   The quality of extracted RNA plays an important role in PCR reactions. The presence of impurities which can be determined by the measurement of absorbance A_{260/280 and} A_260/230 could impact the downstream processing. Also, the primer design is important in the qPCR reaction. It is important to check the melting curve for each gene and confirm it is a single peak. The presence of several peaks indicates poor primer design and the possibility of primer dimer which affects the final interpretation of the results.

Recipes

Table 1: DNase I treatment preparation.

 DNase I is provided as lyophilized powder. Reconstitute it using with DNase/RNase-Free Water.  The volume of added water depends on the unit in DNase I powder. For example, add 275 µl water for 1500 U of DNase I powder and 55 µl water for 250 U of DNase I powder. Mix well and then save the aliquots in -20ᵒC till use.

Table 2: Synthesis of cDNA from RNA using qScript cDNA Synthesis Kit

Table 3: PCR program for synthesis of cDNA using qScript cDNA Synthesis Kit

Table 4: qPCR reaction mixture

^a add ROX Reference Dye 2 (low Conc) to the sybergreen master mix according to the manufacturer instruction to reach to final conc 1x.

^b The amount calculated according to cDNA 500 ng, and dilution is done to 1/5 or 1/10. In general 5-100 ng cDNA/ reaction is acceptable.

C The sequences of primers used in the amplification of inflammatory transcripts in the colon, please refer to Moshensky A et al (Moshensky et al., 2022)

^d Regarding the evaluation of the transcripts of inflammatory cytokines, 10 ul reaction is sufficient since the abundance of these genes in the colon is high. For low abundance genes, it is recommended to use a higher volume reaction system (20-50 ul).

Table 5: PCR program 

Acknowledgments
This study was supported by National Institutes of Health R01HL137052, American Heart Association 16BGIA27790079, University of California, San Diego RS169R, American Thoracic Society Foundation Award for Outstanding Early Career Investigator, and U.S. Department of Veterans Affairs 1I01B x 004767 to Laura E Crotty Alexander. Soumita Das was supported by Tobacco-Related Disease Research Program 28IP-0024.

Competing interests

 The authors declared there is no conflict of interest.

Ethics
This anima study was approved at the University of California San Diego Institutional Animal Care and Use Committee (IACUC protocol S16021). 

References

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Figure 1: Flow of the design experiment

Mice are exposed either to air or Juul pods (Mango or Mint) through the In Expose system. After 3-months of exposure, mice were sacrificed and colon were collected, cleaned from any remaining stool, and then stored in RNA later solution. The colon was either processed immediately or saved at -80C till the processing.                                                                                                             

Figure 2: Steps of RNA isolation from the colon

Colon samples were cut into small pieces, weighted, and then homogenized. The homogenized samples were centrifuged to remove the tissue debris and then the supernatants were processed for RNA extraction. The extracted RNA was used for quantification of the transcripts of inflammatory cytokines.