Optimization of mRNA extraction from human nasal mucosa biopsies for gene expression profile analysis by qRT‐PCR

Quantitative real‐time reverse transcriptase polymerase chain reaction (qRT‐PCR) is the gold‐standard method for analyzing modifications in gene expression in cells and tissues. However, large quantities of high‐quality RNA samples are needed for analyzing the expression of multiple genes from one human tissue sample. Here, we provide an optimized protocol for extracting large amounts of RNA from human nasal mucosal biopsies. The quality and quantity of samples were sufficient for qRT‐PCR analyses of the expressions of various genes, in duplicate. In contrast to other protocols, we optimized RNA isolation to increase the amount from nasal biopsy samples for analyses of multiple genes. In most previous publications, expressions of only one or a few genes, including housekeeping genes, were analyzed because the amount of biological material was small. We were able to improve our protocol with respect to the yield and quality of RNA. This is likely to produce better results from molecular analyses of very small biopsy samples of human nasal mucosa.


| INTRODUCTION
Quantitative reverse-transcription PCR (qRT-PCR) is currently the gold standard for detecting changes in gene expression (Van Guilder et al., 2008). This technique makes it possible to analyze large numbers of genes and samples simultaneously, so it can provide researchers and clinicians with valuable information concerning, for example, tumor development (Sattler et al., 2011).
However, the inaccessibility of human CNS tissue and brain cells in vivo is problematic when qRT-PCR is to be used for predicting or diagnosing neurodegenerative diseases, brain trauma, or psychiatric diseases.
Olfactory loss is increasingly recognized as being associated with reduced quality of life and major health outcomes such as neurodegeneration and death. Trigeminal afferents, as parts of a neuroanatomically independent system, are key to the reception and perception of volatile stimuli (Cain & Murphy, 1980;Doty, 1975).
Biopsy samples of the nasal mucosa could provide an easy-access readout of the molecular background to conditions associated with both olfactory and trigeminal receptor expression. Nevertheless, the amount of material is small and it sometimes has to be divided even further for different analyses. One option for increasing the quality and quantity of material is to pool samples from different nasal regions (e.g., lower and middle meatus) or the two nasal cavities, or from different donors (Poletti et al., 2019). However, this strategy is inapplicable if the sample is to be used for diagnosis or for assessing therapeutic outcomes.
The distribution, regulation, and functions of the abovementioned trigeminal receptors in the nasal mucosa are still not understood. Our group previously attempted to map mRNA densities in trigeminal receptors in various regions of the human nasal mucosa in healthy subjects (Poletti et al., 2019), but the results were not fully consistent with electrophysiological results from a different set of individuals (Meusel et al., 2010).
To understand trigeminal function better, single biopsy samples of nasal mucosa would be very helpful. This entails the challenge of obtaining high-quality RNA from very small samples, but it would elucidate the interplay between olfactory and trigeminal function in patients with loss of smell.
In this pilot study, we provide an optimized protocol for extracting large amounts of RNA from very small human nasal mucosa biopsies.

| Patients and human tissue
The study was approved by the Ethics Committee at the University Clinic of the TU Dresden (application number EK406102018). All participants provided written informed consent. Olfactory testing (using Sniffin Sticks [Oleszkiewicz et al., 2019]) prior to surgery established that the patients had a normal sense of smell. Biopsies were taken during nasal surgery, the patients receiving general anesthesia at the Department of Otorhinolaryngology of the TU Dresden. At the beginning of the operation, approximately 2-3 mm circular mucosal biopsies were taken using Blakesley forceps. The mucosal tissue obtained intraoperatively was immediately stored in RNA-later in a freezer at À20 C and sent on dry ice to the Department of Anatomy, Carl von Ossietzky University of Oldenburg. Samples of healthy human brain tissue (for gDNA control) were taken from one individual in Forensic Medicine at the University Rostock within 10 h of death and stored at À80 C for further study. All procedures were approved by the local Ethics Committee (Rostock University Medical Center; registration ID: A2015-0143). 3. Samples were homogenized using the ball mill shaker 3-4 times for 30 s at maximum frequency.

| Reagents
4. It was important to dip the grinding jar into liquid nitrogen again for 20 s between successive shaking steps. 5. After the last shaking round, 300 μL additional TRIzol were pipetted into the sample and it was then homogenized by pipetting up and down several times. Finally, the homogenized 600 μL sample was transferred to a RNAse-free 1.5 mL Eppendorf cup on ice.
6. Total RNA was extracted following the TRIzol manufacturer's instructions, including a DNAase digestion step before ethanol precipitation (Table 1). Each sample pellet of purified RNA was eluted with 10 μL RNase-free water. 7. RNA concentrations and purity were determined by spectroscopy using an UV-vis spectrometer, and by automated electrophoresis.
2.4.2 | cDNA synthesis and PCR 1. To synthesize cDNA, between 0.05 and 2 μg of total RNA was reverse-transcribed to single-stranded cDNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) following the manufacturer's protocol. The concentration of RNA used depended on the total RNA concentration. Thus, we used 1.2 μg for sample 1, 0.13 μg for sample 2, 0.05 μg for sample 3, 2 μg for sample 4, and 2 μg for sample 5.
2. The quality of the amplified cDNA was tested using beta-actin (Actb) PCR (Figure 1).

| Quantitative PCR
1. Reactions were prepared according to the manufacturer's protocol.
Each sample was diluted 1:10 and 4 μL were used for the qRT-PCR reaction.
T A B L E 1 The exact details of RNA extraction from the five samples, as well as the subsequent quantity and quality controls of the samples. F I G U R E 1 ß-actin PCR from five different human nasal mucosal biopsies (1-5) shows the correct cDNA band at around 490 bp. As a positive control, human genomic DNA was used.
As expected, the ß-actin band appears around 696 bp. The negative control (H 2 O sample) shows no band. As a marker, the hyperladder was used.
2. The following cycling parameters were used for quantitative RT-PCR reactions: 95 C for 20 s, 95 C for 1 s and 60 C for 20 s, for 45 cycles.
3. Duplicates of each sample were used and graphs were produced using the quantification cycle (C q ) value (Figures 2 and 3).

| Genomic DNA extraction
1. Genomic DNA was prepared from human brain tissue according to the manufacturer's protocol (Invitek Molekular).
2. The genomic DNA sample was used as control for contamination of the cDNA samples by ß-actin PCR (Figure 1 and Table 2).

| RESULTS AND DISCUSSION
In this study, we demonstrated an optimized protocol for mRNA extraction from human nasal mucosa biopsies for gene expression profile analysis using qRT-PCR.
The challenge in using human nasal mucosa biopsies is to find the minimal amount of tissue sample necessary to create a reliable map based on trigeminal receptor mRNA.
In comparison to the very large gene family (around 400) of olfactory receptors (OR), the proteins of which are expressed in a very restricted portion of the nasal mucosa, the olfactory cleft (Verbeurgt et al., 2014), only a few trigeminal receptor/channel proteins are widely distributed over the entire nasal mucosa.
We used fresh, unpooled biopsy samples of human nasal mucosa.  (Figure 2). For ß-actin (B), the C q value for new sample 1 was 23.93, compared to 28.07 in the old sample 1 (Figure 2); the C q value for new sample 4 new was 26.58, compared to 28.96 in the old sample 4 (Figure 2). integrity can be measured by the RNA integrity number (RIN). A combination of all three measurements is helpful for evaluating the quality of RNA (Jensen et al., 2021;Shah et al., 2019;White et al., 2018). In our study, the amount of tissue was very small, between 4.0 and 10.9 mg. Nevertheless, we were able to obtain between 26.0 and 812.0 ng of RNA (Table 1). The ß-actin PCR results showed clear cDNA bands in all five samples, with no contamination by genomic DNA (Figure 1), even though the RIN value and the A 260/280 and A 260/230 ratios were very low. In general, RIN >7 is equivalent to "good quality," RIN < 5 to "poor quality." Furthermore, RNA samples are defined as "good-moderate" with an A 260/280 ratio between 1.8 and 2.0 and an A 260/230 ratio between 1.8 and 2.2. An A 260/280 ratio lower than 1.8 means the RNA sample contains proteins. RNA samples with A 260/230 ratios below 1.8 indicate contamination with phenol, TRIzol, etc.
Our samples showed RIN values below 5 (samples 2, 4, and 5) or below detection level (samples 1 and 3) (Table 1). Also, two of the A 260/280 ratio values were below 2.1 (samples 2 and 4), one was below detection level (sample 3), and only two were around 2.0 (samples 2 and 5) ( Table 1). The A 260/230 ratios of all five samples were below 1.8 (Table 1). Nevertheless, the control ß-actin PCR showed clear bands without genomic DNA contamination (Figure 1). Increasing the washing steps reduced the concentration in both samples (Table 3), but increased at least two quality parameters (  Figure 3).
The C q values for Gapdh in sample 1 did not change (from 23.66 to 23.80), but was already in a good range (Shah et al., 2019). For ß-actin, the C q value decreased from 28.07 to 23.93 ( Figure 3)  Several studies have yielded protocols for optimized extraction methods from human or animal tissues (Cepollaro et al., 2018;Nouvel et al., 2021;Ruocco et al., 2017). Depending on tissue handling and storage, preparation is a critical step for any further molecular biological analysis.

| Conclusions
The limitation of this study is the lack of comparison among different extraction methods, but the limited availability of human nasal mucosa biopsies and the very small amount of each sample was a major C q values. Our protocol improved the yield and quality of RNA and should produce better results for molecular analysis in very small human nasal mucosa biopsy samples.

Parts of this study were supported by the Verbund Norddeutscher
Universitäten to A.U.B. and M.W. English-language support was provided by stels-ol.de. Open Access funding enabled and organized by Projekt DEAL.