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, Use a dedicated pH electrode and pH standard solutions, as they can be a source of contamination
, Store borosilicate glass capillaries; 20 µL long, fine and flexible tips; a 20 µL micropipette; a home-made expeller (patch-clamp pipette holder attached to a 10 mL syringe); 500 µL PCR tubes; 10 µL aerosol resistant filter tips; and thin permanent markers in a dedicated box
, During the first PCR, all the genes of interest are co-amplified by mixing together all PCR primers. To detect reliably transcripts from single cells, it is essential to design efficient and selective PCR primers. The use of nested (internal) primers for the second rounds of PCR improves, Primer Design Note: Multiplex RT-PCR relies on two amplification steps
, Retrieve the curated mRNA sequences of the genes of interest in NCBI Reference Sequence Database 32 as well as those of their related family
, Enter the name of the gene(s) and the species of interest as a query and click on the retrieved relevant sequence. 2. To restrict the analysis to the coding sequence of the gene(s), click on Send to (upper right corner), and select Coding sequences and FASTA Nucleotide as format. Then click on Create File and save the FASTA sequence using a
, Multiple Alignment Construction & Analysis Workbench) or any other multiple alignment software to determine the regions of homology. 1. Click on File | New Project... and select DNA as the sequence type. To import the gene sequences, select Sequence | Import Sequence, Use MACAW software, vol.33
, Click and drag all sequences in the Schematic window to select the entire sequences
, Adjust the Pairwise score cutoff to a relatively high value (e.g., 1,000) and the Min. seqs. per block to the total number of sequences to align and click on Begin
, In the appearing Search Result window, select the result(s) with the highest MP-score and click on Link. If no result is found, decrease the Pairwise score cutoff and/or the Min. seqs
, To facilitate the visualization of homologous regions, select Alignment | Shading | Mean score
, To obtain the most specific primers, select the regions with the least sequence homology
, Fetch the sequences of all splice variants and repeat steps 2.2.1-2.2.6. Select their common regions for an overall detection. Consider alternative cassettes for dedicated splice variants analyses 20, vol.34, p.36
, Align the mRNA sequences on the animal model genome using BLAST 37 genomes (Basic Local Alignment Search Tool) to determine the intron-exon structure of the genes
, Select the BLAST mouse genome by clicking on Mouse and upload the retrieved FASTA sequence in the Enter Query Sequence section. In the Program Selection, select Optimize for Highly similar sequences (megablast)
, Make sure that it corresponds to the gene of interest as indicated in features of the Alignments section
, Sort the alignment by Query start position in the same section to obtain the exons succession of the coding sequence. Note the start and end positions of all hits which roughly correspond to the position of the introns on the query sequence
, In case of intron-less genes, the collection of the nucleus is problematic as it can lead to potentially confounding results. To address this issue, include a set of primers aimed at amplifying an intronic sequence to probe for the presence of gDNA 25
, Journal of Visualized Experiments, vol.136, p.12, 2018.
, To achieve an efficient amplification, select primers generating amplicons with a length ideally comprised between 200 and 400 base pairs
, To amplify simultaneously various cDNAs during the multiplex PCR step, design primers with a length between 18 and 24 nucleotides and a melting temperature (Tm) between 55 and 60 °C
, To minimize the formation of secondary structures, which reduce the amplification yield, select sense and anti-sense primers with minimal hairpin and duplex lengths (Figure 2B)
, Design internal primers using the same criteria (steps 2.5-2.8)
, Verify the specificity of the PCR primers by aligning the primers on the Reference RNA Sequence Database of the organism of interest using a nucleotide BLAST
, BLAST
, In the Choose Search Set section, click on Others (nr etc.), select Reference RNA sequences (refseq_rna) in the Database option and specify the organism (e.g., Mus musculus (taxid:10090)) to restrict the analysis to the desired species
, In the Algorithm parameters section reduce the word size down to 7 to increase the chance to obtain hits
, To design selective primers, keep only those whose sequence has at least 3 mismatches with undesired cDNAs when possible
, µM) to ensure that all external primers can be mixed together at a final concentration of 1 µM, Order desalted primers at a relatively high concentration
, Preparation of RT Reagents 1. 5x RT-mix: Prepare in RNase-free water 400 µL of working RT mix solution (5x) containing random primers at 25 µM and dNTPs at 2.5 mM each
, Prepare 1 mL of 0.2 M DTT in RNase-free water and store it as 50 µL aliquots
, Store 2,500 U of RNase inhibitor (40 U/µL) and 10,000 U of reverse transcriptase (RTase, 200 U/µL) as 5 µL aliquots
, To ensure an optimal quality of the RT reagents, store the aliquots at -80 °C. Use them for up to 10 cells and only for one day
, PCR Validation 1. Prepare cDNAs by doing a reverse transcription on a relatively large amount of total RNA (typically 1 µg) extracted 38 from the structure of interest. 1. Do not use the pipettes dedicated to single cell RT-PCR for these preliminary steps but use the RNase-free pipettes
, In a 500 µL PCR tube, add 1 µL of diluted total RNA and 8 µL of RNase-free water. Denature at 95 °C for 1 min and cool down the tube on ice
, Add 4 µL of RT 5x buffer supplied by the manufacturer, 4 µL of RT mix solution, 1 µL of RTase, 1 µL of RNase inhibitor, and 1 µL of 200 mM DTT
, Flick the tube, spin it, and incubate overnight at 37 °C
, Store the cDNAs at -80 °C for up to several years. Prepare an aliquot of cDNAs diluted to 1 ng/µL of total RNAs equivalents
, Mix and dilute each primer pair at 1 µM with the pipettes dedicated to single cell RT-PCR. Use 20 µL of each primer mix for 100 µL PCRs
, On ice, prepare for each primer pair a premix containing: water (q.s., 80 µL), 10 µL of 10X buffer, 1 µL of 100x dNTPs, vol.50, pp.500-501
, Add 2 drops (~100 µL) of mineral oil to the premix without touching the wall or the cap of the PCR tube
, To minimize the formation of primer-dimers, perform a hot start by placing the PCR tubes in the thermocycler pre-heated at 95 °C. After 30 s, quickly expel 20 µL of the primer mix on top of the oil
, After 3 min at 95 °C, run 40 cycles (95 °C, 30 s; 60 °C, 30 s; 72 °C, 35 s) followed by a final elongation step at 72 °C for 5 min
, Analyze 10 µL of each PCR products by agarose gel electrophoresis (2%, weight/volume) 39 . If some PCR products do not have the expected size, re-design other primers (see Section 2)
, Consult the local safety office before using it. Always wear gloves when manipulating it. Use a commercially available solution of ethidium bromide (10 mg/mL) solution instead of powder to minimize the risk of inhalation. Similarly, UV light can be harmful, so make sure to wear UV protection safety glasses or a mask
, Once all primer pairs have been individually validated, test the multiplex protocol
, Mix and dilute all external primers together at 1 µM. Store the multiplex primers mix at -20 °C for up to several weeks. 2. On ice, prepare a premix containing: water (q.s., 80 µL), 10 µL of 10X buffer, 1 µL of 100x dNTPs (50 µM of each), 500-1,000 pg of cDNAs diluted at 1 ng/µL
, Flick the PCR tube, spin it, and add two drops (~100 µL) of mineral oil
, Adjust all the volumes according to the number of genes to analyze and consider using 10% of extra volume to compensate for pipetting errors
, Shake gently, spin the tube, dispatch 80 µL of premix in each PCR tube, and add two drops (~100 µL) of mineral oil per tube without touching the wall or the cap of the tubes
, For 60 mL of internal patch-clamp recording solution
,
, Add 40 mL RNase-free water and dissolve 2.02 g of K-gluconate (144 mM final, 143 mg of HEPES (10 mM final), and 180 µL of 1 M MgCl, vol.2
, Since the internal solution is also used as a buffer for the reverse transcription, carefully control Mg 2+ concentration. Compensate for a lower Mg 2+ concentration in the internal solution by adding MgCl 2 afterwards to reach a final concentration of 2 mM in the RT reaction
, To label the harvested cell after patch-clamp recording, add 2-5 mg/mL of RNase-free biocytin to the internal solution described above
, 22 µm pore size) the internal solution and store it at -80 °C as 100-250 µL aliquots
, To validate the use of the internal solution for single-cell RT-PCR, add 0.5 µL of total RNAs diluted at 1 ng/µL to 6 µL of patch-clamp solution and leave it on the bench for about 30 min
, With a 2 µL micropipette, add 2 µL of 5x RT-mix, 0.5 µL of 20x DDT, 0.5 µL RNase inhibitor, and 0.5 µL RTase, and incubate overnight at 37°C
, On ice add water (q.s., 80 µL), 10 µL of 10X buffer
, Note: Omitting the 0.5 µL of total RNA and replacing it with 0.5 µL RNase-free water will ensure that the internal solution is free of RNA and/ or DNA contaminations
, Acute Slice Preparation Note: This protocol describes the slicing procedure for juvenile (i.e., less than 28 postnatal days) male and female mice
, Prepare 2 L of aCSF containing (in mM) 125 NaCl, 2.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 10 Glucose, and 15 Sucrose. To reduce glutamatergic activity during slice preparation, prepare a cutting solution by adding 1 mM of kynurenic acid
, Before slicing, prepare a dissection kit containing surgical scissors, fine iris scissors, two spatulas, forceps, a disc of paper filter, and cyanoacrylate glue
, Use ice-cold cutting solution saturated with O 2 /CO 2 (95%/5%) and cool down the cutting chamber at -20 °C
, Anesthetize the mouse with a small paper towel soaked with isoflurane. After ~2 min, make sure that the mouse is under deep anesthesia by verifying the absence of response to paw pinch
, Quickly decapitate the mouse. Remove the scalp and open the skull. Extract the brain carefully and place it into a small beaker filled with ice cold (~4 °C) cutting solution oxygenated with O 2 /CO 2
, Remove the cutting chamber from -20 °C conditions and remove moisture with a paper towel
, Carefully dissect the brain to isolate the region of interest, glue it on the cutting chamber, and add ice-cold cutting solution oxygenated with O 2 /CO 2
, Transfer them at room temperature in a resting chamber filled with oxygenated cutting solution and allow them to recover for at least 0, vol.5
, Single Cell RT-PCR after Patch-clamp Recording 1. Clean the perfusion system regularly with ~100 mL of 30% H 2 O 2 solution and rinse extensively with ~500 mL of distilled water to avoid bacteria growth, as it is an overlooked source of RNase contamination
, Chlorinate the filament with a patch pipette filled with concentrated bleach every harvesting day. Do not use a micropipette to fill the patch pipette but rather a 20 µL long, fine, flexible tip attached to a 1 mL syringe. Then, rinse it extensively with RNase free water and dry it with a gas duster
, Prepare a small box of ice containing 5x RT-mix and 20x DTT aliquots. Store the RTase and RNase inhibitor aliquots at -20 °C in a benchtop cooler
, -2 µm open tip diameter, 3-5 M?) from borosilicate glass while wearing gloves and fill one of them with 8 ?L of internal solution, Pull patch pipettes
, Place the pipette in the pipette holder while wearing new gloves and not touching the filament with fingers
, Approach the patch pipette with a positive pressure and perform whole-cell recording to characterize the targeted cell
, At the end of the recording, prepare a 500 µL PCR tube filled with 2 µL of 5x RT Mix and 0.5 µL of 20x DTT, spin it, and store it on ice
, Journal of Visualized Experiments, vol.136, p.12, 2018.
, Avoid as much as possible collecting the nucleus if some intron-less genes are considered. In such a case always include a set of primers aimed at amplifying gDNA to probe for genomic contamination
, Withdraw the pipette gently to form an outside-out patch to limit contamination by the extracellular debris (Figure 4) and to favor the closure of the cell membrane for subsequent histochemical analysis
, Keep in mind that knobs, micromanipulators, computer keyboard, or computer mouse are potential sources of RNase contamination
, Attach the pipette to the expeller and expel its content into the PCR tube by applying a positive pressure
, Break the tip of the pipette into the PCR tube to help the collection of its content
, Briefly centrifuge the tube, add 0.5 µL of RNase inhibitor and 0.5 µL of RTase, mix gently, centrifuge again, and incubate overnight at 37 °C. 17. Spin the tube and store it at -80 °C for up to several months until PCR analysis
, Perform the first amplification step directly in the tube containing the ~10 µL of RT products by adding water (q.s., 80 µL), 10 µL of 10x buffer
, Histochemical Staining of the Recorded Cell
, Following the electrophysiological recordings, maintain the slice for 20 min in oxygenated aCSF to allow the diffusion of biocytin in the axonal and dendritic tree
,
, Wash the slices 4 times, 5 min each, with 1 mL Phosphate Buffered Saline (PBS)
, In the same well, permeabilize and saturate the slice during 1 h at room temperature with 1 mL of PBS supplemented with 0.25% Triton X-100 and 0
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