Fluorescent RNA random primer-triggered polymerase chain reaction assay
Differential display polymerase chain reaction (PCR) can facilitate the identification of new molecular markers associated with contamination exposure in many species. To date, several differential display methods have been described in detail. Here, we describe a modified RNAarbitrarily primed PCR (RAP-PCR) method that involves fluorescent labeling of cDNA transcripts by rhodamine-labeled 18-base random primers. These random primers bind specifically to the coding region of the cDNA, thus simplifying the downstream identification of toxicant-responsive genes. This method has been named fluorescent RNA arbitrarily primed PCR (FRP), or FRNAP.
This method, named fluorescent RNA arbitrarily primed PCR (FRAP-PCR), has been successfully used for several avian species and for RNA from cultured cells and tissues, etc. This direct, safe, and low-cost method is a valid alternative to the radiolabel-based RAP-PCR method.
By Martin, this experiment is from "Environmental Genomics Lab Guide".
Operation method
Fluorescent RNA random primer-triggered polymerase chain reaction experiments Move ## I. Isolation of RNA from tissues and cultured cells Caveat 1. Cell culture should be performed under strict aseptic conditions. Immediately after removal of the medium, the plates should be placed on dry ice or placed at 1 80°C until RNA is extracted. Tissue must be collected using sterile, RNase-free instruments treated with 3 % hydrogen peroxide. Tissue samples should be rapidly frozen in dry ice or liquid nitrogen to prevent degradation of the RNA. Alternatively, the tissue can be cut into small pieces less than 0.5 cm in diameter and stored in RNAlater (0.1-0.2 g/mL, Ambion) at 4°C for up to one month or at _20°C for long-term storage. This is ideal for on-site sampling, eliminating the need for dry ice or liquid nitrogen. 2. Removal of trace amounts of chromosomal DN A or other sources of genomic DNA contamination is critical to the success of FRAP-PCR experiments. Genomic DNA can compete with cDNA during the amplification reaction leading to false positive results in RNA fingerprinting. Studying these false positives is a waste of reagents and time. We use Ambion's DNA-Free K it to remove single- and double-stranded genomic DNA while maintaining the integrity of the RNA template. The non-transcription control in the subsequent real-time quantitative PCR reaction did not show any amplification products. An additional advantage of this kit is that it contains a new DNase removal reagent that removes DNase and divalent cations to eliminate their effect on subsequent reactions. The DNase and divalent cation removal procedure is very rapid and does not require organic solvent extraction, addition of EDTA, or heat inactivation (all of which may affect the integrity of the RNA). 3. Dispense the appropriate amount of RNA per tube to avoid repeated freezing and thawing of RNA for subsequent cDNA synthesis reactions, which may affect the quantity and quality of RNA. Repeated freezing and thawing of the total RNA sample will reduce the brightness of the bands on gel electrophoresis. 4. The optimal amount of total RNA for FRAP-PCR experiments was obtained by pre-testing the results with 200, 500, 750 and 1000 ng. The 750 ng results were the most reproducible with the clearest bands. However, higher noise-to-signal ratios are sometimes encountered, and lowering the total amount of RNA to 375 ng eliminates background noise and improves the clarity of the bands. 5. Depending on the lysate and the type of tissue being lysed, we used both sterling steel beads and tungsten carbide beads to homogenize the tissue. We found that TRIzoI corrodes stainless steel beads while RNeasy lysate (Qiagen) does not. Gonadal, heart and liver tissues dissociated better with tungsten carbide beads, while brain and thyroid tissues dissociated better with stainless steel beads. 6. The reason for cDNA partitioning is the same as the reason for RNA partitioning: to avoid degradation caused by repeated freezing and thawing. If the cDNA obtained from a reaction system of 20 is used in 4 FRAP-PCR reactions, it will be freeze-thawed 3 times in the middle, and the third time it will affect the gel pattern, especially the weak bands. 7. After first-strand synthesis, a low-fidelity PCR step is performed to facilitate the binding of random primers to the ends of the PCR product. This second-strand synthesis step ensures optimal amplification of the cDNA product in the subsequent high-fidelity PCR. 8. It is important to determine the optimal recovery temperature for the specific 18-base primers used in FRAP-PCR. The ideal annealing temperature for A3 and B3 is 54°C, and for C3 it is 56°C. If a researcher uses primers other than those described in this chapter, it may be necessary to use an annealing temperature that is different from the one described in this chapter. If the researcher is using 18-base primers other than those described in this chapter, the dissolution temperature, the GC content, and the optimal PCR temperature need to be determined using analytical software (e.g., OligoanaIyzer) prior to performing the experiment. Afterwards, PCR conditions are determined empirically. 9. In the initial stage of setting up the FRAP-PCR method, the rhodamine-labeled PCR product was not purified prior to electrophoresis. This resulted in large non-specific bands in all wells (Fig. 2a). We hypothesized that the non-specific bands were due to excess nucleotides and excess rhodamine. The QIAquick PCR Purification Kit (Qiagen) recovers fragments from 100 bp to 10 kb and removes fragments smaller than 40 bp. This simple and fast column-based method is very effective in removing tailing and improving the resolution of electrophoresis results (Figure 2b) and should be used for all FRAP-PCR experiments. In unpurified samples, background due to non-specific bands can lead to loss of the target band. 10. Samples should be loaded without the 5 wells as close to the sides as possible. Electrophoresis results from these wells often show arching bands, which make it difficult to compare the results with those from the center wells. This makes it difficult to compare the results with those of the center wells, because the center wells usually have better separation effect and the electrophoretic bands are clear and flat. At the same time, opening 1~2 sample wells between different treatment groups can avoid the dispersion interference of electrophoresis bands. 11. The rhodamine-labeled DNA standard was not run on the sequencing gel at the same time, therefore, the product size was carried out in the re-amplification PCR step. After 6 h of constant voltage electrophoresis at 1700 V, the size of the product cut in the middle of the sequencing gel is about 300~750 bp. In most cases, the bands cut from the sequencing gel yielded a single product during re-amplification PCR, which could be subcloned directly into the TOPO vector. However, some bands that appear to represent a single product will show multiple bands on agarose gels used for screening. This phenomenon of co-mobility and non-specific amplification can be addressed by either (1) cutting electrophoretic bands from the agarose gel that are the same size as the bands on the sequencing gel, purifying them with the QIAquick PCR Purification Kit (Qiagen), and subcloning the purified product, or (2) increasing the time of electrophoresis of the sequencing gel in order to improve the resolution of the individual bands, since it is possible that the original bands represent more than one PCR product. 12. Depending on the number of potential targets to be further investigated, -transformation and cloning may represent a significant proportion of the experimental cost of FRAP-PCR. We found that cloning reactions can be performed with half the recommended amount of reagents. For example, only 0.5 of the vector was used in the ligation reaction, and the ligation product was transformed into a human half-tube (25 juL) of sensory bacteria. This saves reagents and costs without affecting the efficiency of TOPOTA cloning. 12. There are several methods for identifying differential gene expression after cloning. One of the most sensitive and reliable methods for measuring the relative expression of mRNA, real-time quantitative RT-PCR (MX4000, SYBR GREEN Master M ix fluorescent dye), was chosen. The primers for real-time quantitative PCR were designed according to the sequences obtained by FRAP-PCR. 18SrRN A, ␣-actin, and glycerol-3-acid dehydrogenase (GAPDH) were used as internal control genes to compare with the expression abundance of target genes. For more product details, please visit Aladdin Scientific website.
1. TRIzol reagent (Invitrogen). This reagent contains phenol and thiocyanate compounds, and should be handled in solid cotton.
The reagent contains phenol and thiocyanate compounds and should be handled in lab coat and gloves and stored at 4°C.
2. Chloroform.
3. Isopropyl alcohol.
4. Ethanol.
5. diethyl pyrocarbonate (DEPC) treated water.
6. DNA-free kit (Ambion). Store at 20°C.
##CDNA Synthesis
1. Superscript™ II, RNase H-reverse transcriptase (200 U/(nL; Invitrogen). Stable at 20°C.
can be stored stably at 20°C for more than two years.
2.5X First Strand Synthesis Buffer: 250 mmol/L Tris-HCI, pH 8.3, 375 mmol/L KCl, 15°C, 1.5 μg/kg, 1.5 μg/kg, 1.5 μg/kg, 1.5 μg/kg.
L MgCl2, stored at 20°C. The buffer was used for the synthesis of the first strand. The first-strand synthesis buffer.
3.0. lmmol/L dithiothreitol (DTT), stored at 20°C.
4. dNTP mixture: dNTP 10 mmol/L each, stored at 20°C. 4. dNTP mixture: dNTP 1 0 mmol/L each, stored at 20°C.
5.18 Random primers for bases (25 pmol/L): A 3, 5'-AATCATGAGCTCTCCTGG--3'; B3, 5'-CATACACGCGTATACTGG--3'; C3 , 5'-CCATGCGCATGCATGAGA- 3 : stored in a 20.
6.RNaseOMT™ Recombinant Ribonuclease Inhibitor UOU/^L ; Invitrogen), I t is stored at 20 °C.
## III. FRAP-PCR
1. QiagenT called DNA polymerase (5 U/|uL; Qiagen), |^; store at 20°C.
2. Qiagen IOXPCR buffer: Tris-HCl, KCl, (NH4)2SO4, 15 mmol/L MgCl2, p H 8.7, C stored at 20°C .
3.25 mmol L MgCl2, I t was stored at 20°C .
4. Diisopropyl pyrocarbonate (DEPC)-treated water.
5. dNTP mixture: 10 mmoL/L each of dNTP, JC stored at 20°C.
6. Random primers (lOfxmol/L; A3, B3, or C3) labeled with rhodamine at the 5' end, stored at 20°C, protected from light.
7. QIAquick PCR Purification Kit (Qiagen)
##Gel electrophoresis
1. Gel preparation solution: 6 % acrylamide (29: 1 acrylamide-bisacrylamide), containing 7 mol/L urea and 1XTBE buffer.
2.1 parent-butyl 6£ buffer: 89 1^11 〇 1, 1 1 1 ^ alkali, 89 111111 〇 1/1 ^ boric acid, 2 111111 〇 1 / ethyl£ 0 butyl octasodium
Salt, pH 8. 3.
3. Sampling buffer: 9 9 % formamide, I mmol/L EDTA, pH 8.0, 〇. 0.009% bromophenol blue, 0.009% xylene cyanide.
4.TEMED.
5.25% ammonium persulfate.
6. FDD Vertical Electrophoresis System with 60-well comb and slides with low background fluorescence (GenHunter).
7. Sigmacote 桂胶液（Sigma-Aldrich）， JC stored at 4°C。
8. DC power supply >1700 V available.
9. Burst Light Imaging Scanner: Typhoon 9210 Variable Mode Imager (Amersham).
10. Inkjet printer capable of printing llin X 17in® & for printing actual size gel images.
11. Blades.
##V. Isolation, re-amplification and cloning of PCR products
1. Gelatin (20 ZmL), stored at _20°C.
2.3 mol/L sodium acetate.
3. ethanol.
4. diethyl pyrocarbonate (DEPC) treated water.
5. PCR reagents, see 2.3 (Note: lOfxmol/L unlabeled random primers).
6. TOPOTA® Cloning Kit (Invitrogen). OneShot® Sensitive E. coli are stored at -80°C and the PCR TOPO® Cloning Vector is stored at 1-2 CTC.
7. SOC medium: 2 % tryptone, 0.5 % yeast extract, 10 mm SOP. 5 % yeast extract, 10 mmol/L NaCl, 2.5 mmol/L KC1, 10 mmol/L MgCl2, 10 mmol/L MgSO4, 20 mmolZL glucose.
8.L B medium.
9. ampicillin (10 mg/mL), JC stored at 1 20°C .
10. Agar.
11. X-gal (20 mg/mL), stored at _200 °C, protected from light.
12.M13 primer: forward (5'-GTAAAACGACGGCCA0 3'), reverse (5'-CAGGAAACAGCTATGAC-3'), stored at 1-20°C.
13. QIAprep Centrifugal Small Volume Plasmid Extraction Kit (Qiagen) 3.
## VI. RNA extraction from tissues or cultured cells
RNA can be isolated from a variety of sources including cultured cells (e.g. neuronal cells, hepatocytes, etc.) and tissues (see Note 1). Purification of mRNA is not necessary for the FRAP-PCR method because mRNA represents only 3 % to 5 % of the total cellular RNA (13). Thus, the methods described in this chapter are suitable for situations where the total RNA available is limited (e.g., small amounts of cells or tissues). Total cellular RNA is easy to purify but needs to be treated with DNase before use in FRAP-PCR (see Note 2) and dispensed in disposable tubes according to the amount used each time (see Note 3). The amount of total RNA used for each experiment is approximately 750 ng (see Note 4).
1. Primary cells (~1.2 mg) from each well of the culture plate are lysed with 100 A TRIzoI, or I mL TRIzol per 50-100 mg of tissue, and homogenized with a homogenizer Mixer Mill MM 300 (Retsch) and Crane Carbide alloy beads at 20 Hz for 2 min (see Note 5).
2. Allow the homogenized sample to stand at room temperature for 5 min to allow complete dissociation of the nucleoprotein complex.
3. Add 0.2 mL of chloroform to each I mL of TRIzo, shake the tube vigorously by hand for 15 s, and let stand at room temperature for 2~3 min.
4. Centrifuge at 12 000 g at 4°C for 15 min.
5. Transfer the upper aqueous phase (about 60% of the starting TRIzol volume) to a new tube and add 0.5 mL of isopropanol per Im L of TRIzol, mixing several times by inversion. Allow to stand at room temperature for 10 min, then centrifuge at 4°C for 20 min at 12 OOOg.
6. Remove the supernatant, wash the RNA precipitate with I mL of 75% ethanol per I mL of TRIzol, mix well with shaking, and centrifuge at 7500 g for 5 min at 4°C.
7. Dry the RNA at room temperature and dissolve in <100 fxL of DEPC-treated water.
8. Determine the concentration of RNA using a spectrophotometer.
9. To decontaminate genomic D N A, add 5 (uLDNase I buffer and I fxL recombinant DNase I (2 UZfjtL) per 1 0 鸿 R N A in a 50 reaction system. Incubate at 37°C for 20-30 min.
10. Add 10 juL of DNase I inactivator and leave at room temperature for 2 min, then centrifuge at 10,000 g for I.5 min and transfer the RNA into a new centrifuge tube.
11. Re-measure the RNA concentration, dilute to 75 ng/fx L, dispense IOfL per tube and store at 80°C.
##CDNA Synthesis
In this technique, an 18-base random primer was used instead of a 0 lig0-d T primer to ensure that the synthesized reverse transcription product was not biased toward the 3' end of the untranslated region. This allows R N A sequences that include readable frames internally to be reverse transcribed into cDNAs, thus facilitating the next step of gene product identification. This is of particular interest for species with unknown genome sequences (e.g., silver gulls, mallards, etc.). At the same time, it is necessary to set up a control without reverse transcriptase to determine whether there is genomic DNA contamination in the subsequent steps of the PCR experiment (non-reverse transcription control, no RTcontro丨). In addition, commercially available RNA (e.g., total RNA from transformed rat fibroblasts without DNA contamination, GenHunter) can be used as a control for reverse transcription-dependent mRNA amplification reactions (control RNA).
1. Mix 10 fzL of 75 ng/juL total RNA and I dNTP mixture, I /xL random primer (A 3, B3, or 〇 3; 25_○ 1/b). Incubate at 65°(: for 5 111 丨 11 , place immediately in an ice bath and centrifuge slightly.
2. Add 4 pL of 5X First Strand Synthesis Buffer, 2 DTT, I pL of R N A enzyme inhibitor RNase Out and I /xL of Superscript™ ]! Reverse Transcriptase.
3. Incubate at 25°C for 10 min and then at 42°C for 50 min.
4. Heat at 70°C for 15 m in to terminate the reaction.
5. cDNA is divided into 5 tubes and stored at 1-20°C for subsequent PCR reaction (see Note 6).
##FRAP-PCR
Rhodamine is a photosensitive dye, so all procedures, including primers, working solutions and PCR products containing fluorescent markers, should be performed in the dark.
1. Each 25 fxL reaction system should consist of: 5 pL of cDNA, 2.5 fiL of 10X PCR buffer, 1.5 fJL of MgCl2, 0-5 fJL of dNTP mixture, 1.25 ML of rhodamine-labeled primer (A3, B3, or C3), 0.I fxL of Qiagen Tag DNA polymerase, 14 15 pL of DEPC-treated DNA polymerase, and 1.5 fJL of PCR products. pL DEPC-treated water.
2.PCR parameter settings:
3. Store PCR products at 4°C in a dark place protected from light.
4. The PCR product was purified using the Qiagen PCR clean-up kit (QIAquick Nucleotide Removal kit) (see Note 9).
5. Add 125 vials of PB buffer to 25 x L of PCR product and mix well.
6. Place the QIAquick column in a 2-mL collection tube, add the homogenized sample to the column, and centrifuge at 17,900g for 30-60 s. The QIAquick column is then filled with the PCR product (see Note 9).
7. Discard the liquid from the column and place the purification column back into the collection tube.
8. Add 0-75 mL of PE buffer to the purification column and centrifuge for 30-60 seconds.
9. Discard the filtrate and return the purification column to the collection tube and centrifuge for another Im in to remove the residual buffer.
10. Place the purification column in another clean 5 mL centrifuge tube.
11. Add 30 fxL of EB buffer to the middle of the membrane of the column and allow to stand at room temperature for Im in, then centrifuge to elute the DNA.
##Gel electrophoresis
1. Wipe the inner surface of the slide clean and treat the inner surface of the slide with the grooves with the liquid.
Assemble the slide into a "sandwich" and seal the edges to prevent leakage of the gel before polymerization.
2. Prepare a 6% denaturing polyacrylamide gel. The vertical swimming system (length 45 cm, width 25 cm) requires about 50 mL of polyacrylamide solution. Add 1OO of freshly prepared ammonium persulfate and 50/xL TEMED and mix well.
3. Add the configured gel solution along the top edge of the slide, insert a spiking comb and allow it to polymerize overnight. Cover the top of the gel solution with a damp paper towel and plastic wrap to prevent the solution from evaporating and drying out.
4. After polymerization, pre-electrophoresis is performed for 45 min at 1500 V. A total of about 750 mL of IX TBE electrophoresis buffer is required for the upper and lower buffer tanks.
5. Mix 30/iL of PCR product with 15 FDD of sample buffer and set up appropriate controls (non-transcription control and control for reverse transcription-dependent mRNA amplification) and incubate at 80°C for 2 min before sampling with 5 juL of sample (see Note 10). Before sampling, flush each well with a pipette. Sample three parallel wells per sample.
6. After 6 h of electrophoresis at 1700 V, remove the gel and scan with a Typhoon Imager (excitation lens 532 nm, emission lens 580 nm BP30) according to the instructions for rhodamine. Ensure that the control well lane for the non-transcription reaction is blank and that the control lane for the reverse transcription-dependent mRNA amplification reaction shows a band.
7. Print out the gel pattern according to the actual size, place the gel on the printed pattern and wrap the whole gel with plastic wrap to prevent water evaporation.
8. Cut off the band of interest with a clean blade and rescan to make sure the correct band is being cut. The cut bands should be displayed in the presence/absence mode (Figure 1).
##X. PCR Product Isolation, Re-amplification and Cloning
1. Place the cut strips in a I.5 m L centrifuge tube and soak in 100 bucket of water for 10 min at room temperature.
2. Fasten the lid of the centrifuge tube, incubate at >95°C for 15 min, and centrifuge for 2 min.
3. Transfer the supernatant to another centrifuge tube; add IOyL of 3 mol/L sodium acetate, 2.5 fxL of collagen QOjl ^ ^ ^ L) and 450 yL of anhydrous ethanol. Allow to stand at 80°C for 30 minutes or more.
4. Centrifuge for 10 min to precipitate the DNA, remove the supernatant, rinse the precipitate with 200 ° 85 % ice ethanol, and centrifuge to remove the residual ethanol.
5. Dissolve the precipitate with 1ug of DEPC water and take 4uL for re-amplification. Except for the unlabeled primer (10umol/L), the same PCR reagents as FRAP-PCR should be used for the re-amplification reaction. The PCR cycling conditions were as follows: 94°C, 30 s; 54°C, l min; 72°C, Imin; 30 cycles.
6. 15 PCR reactions were electrophoresed on a 1 % agarose gel and stained with ethidium bromide to determine the size of the insert fragments (see Note 11).
7. Clone the PCR product into the pCR2.1 TOPO® vector using the TOPO® TAB Cloning Kit (Inivtrogen) as follows: 2 p i PCR product, 0 -5 FL pCR2.I TOPO® vector, 0.5 salt solution (see Note 12).
8. Incubate at room temperature for 5 min, store at 120 °C or proceed directly to transfection (steps 9 to 12).
9. Gently mix 2 ligations with 25 tubes of One Shot® Receptor E. coli (about half a tube) (do not pipette the mixture).
10. Ice bath for 5 to 30 min. Heat at 42°C for 30 s, transfer to ice immediately.
11. Add 125 juL of SOC medium and incubate for I h at 37°C on a horizontal shaker at 200 r/min.
12. Spread 50-75 fxL of transformants on LB agar plates containing 50 ⁄^L ampicillin and 40 fxg/mL X-gal, and incubate at 37°C overnight.
13. Touch the edges of the three white clones with the tip of a pipette gun, dissolve each of them in 50 water, and take I to perform a PCR reaction with the M13 primer to identify the positive clones.
14. Select one positive transformant clone from each band of interest and incubate overnight at 37°C in LB/benzylamine medium.
15. Plasmids were extracted and sequenced using Qiagen's QIAprep Small Volume Plasmid Extraction Kit.
16. Verify the expression of different genes (see Note 13). 
