PCR-based Digoxigenin-labeled RNA probe synthesis and in situ hybridization on paraffin-embedded E15.5 murine kidney (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Edward Daniel; Simon Lee; Ondine Cleaver

Keywords

[‘kidney’, ‘in situ hybridization’, ‘probe synthesis’, ‘pcr’]

Subjects

[‘Developmental biology’, ‘Molecular biology’, ‘Gene expression analysis’]

Release Date

2016-06-02

Abstract

This protocol has been optimized for high-throughput, rapid analysis of gene expression of multiple candidates using sectioned embryonic mouse kidneys. Protocols covers all steps from probe generation, tissue isolation and preparation, to expression analysis. Probe generation has been optimized to generate multiple probes inexpensively from cDNA. Protocol can be carried out in a week, and halted as desired at multiple different points.

Reagents

• E15.5 embryonic kidneys for sections
• E18.5 embryonic kidneys for cDNA synthesis
• Sterile PBS
• QIAShredder columns (Qiagen, cat no. 79654)
• DEPC-Treated Water
• Qiagen RNeasy Mini kit (Qiagen, cat no. 74104)
• SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, cat no. 11904-018)
• Gene-specific primers with T7 promoter
• GoTaq Green Master Mix, 2x (Promega, M712C)
• UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Thermofisher Scientific, cat no. 15593031)
• RNAse-free tubes
• PCR tubes
• Chloroform
• 3M Sodium Acetate (eg. Fisher Scientific, cat no. BP333-1)
• 5mg/mL Glycogen (Ambion, cat no. AM9510)
• Ethanol
• dH2O
• MEGAscript T7 Transcription Kit (Thermofisher Scientific, cat no. AM1334)
• Anti-digoxigenin-AP, Fab fragments (Roche, cat no. 11093274910)
• Micro Bio-Spin™ P-30 Gel Columns, Tris Buffer (RNase-free) (Biorad, cat no. 7326250)
• Agarose
• Loading Dye
• 5M Sodium Chloride and anhydrous Sodium Chloride
• Tris-HCl pH 7.4
• Calcium Chloride
• Glycerol
• Sodium Citrate
• SDS
• Heparin
• Formamide
• Maleic Acid
• Tween-20 (Sigma, cat no. P9416-50)
• 2M Tris, pH 9.5
• 1M Magnesium Chloride
• Sodium Hydroxide
• Blocking reagent (Roche, through Sigma, cat no. 11096176001)
• Xylene
• BM Purple (Roche, cat no. 11442074001)
• Proteinase K, anhydrous (Fisher Scientific, cat no. BP1700-100)
• Paraformaldehyde
• CAS-Block Histochemical reagent (Invitrogen, cat no. 008120)
• Rat anti-PECAM antibody (MEC 13.3, BD Pharmigen, cat no. 553370)
• Rat anti-Endomucin antibody (V.7C7, Santa Cruz, cat no. sc-65495)
• Anti-rat 488 antibody
• ProLong Gold antifade reagent (Life Technologies, cat no. P36930)
• Nail polish

Reagent Setup

• Proteinase K stock concentration is 20 mg/mL. Resuspend 20 mg of Proteinase K in 1 mL of the following solution and store at 4oC (Note: these are the FINAL concentrations for each reagent):
  • 20 mM Tris-HCl pH 7.4
  • 1 mM CaCl
  • 50% Glycerol

All solutions below should be filtered using a vacuum and filter paper once all ingredients have been fully mixed together.

• 20x SSC Solution recipe:
  • 87.65g NaCl
  • 44.1g Na Citrate
  • dH2O to 500 mL
  • pH to 4.5
• Pre-hybridization buffer recipe:
  • 500 uL formamide
  • 500 uL 5X SSC pH4.5 (diluted down from above in dH¬2O)
  • 1% SDS – warmed to a clear solution
  • 50 ug/mL heparin
• 5x MBST Solution recipe:
  • In 600 mL dH2O, mix:
    • 150 mL 5M NaCl
    • 58.035g Maleic Acid
    • 5 mL Tween-20
  • Add NaOH to raise pH to 7.5
  • Bring up volume to 1 L
  • Store at 4oC
• NTMT Solution:
  • 20 mL 5M NaCl
  • 50 mL 2M Tris pH9.5
  • 50 mL 1M MgCl2
  • 1 mL Tween-20
  • 879 mL dH2O

Equipment

Equipment

• Dissecting scope
• Black dissection dishes (Living Systems Instrumentation, DD-50-S-BLK-3PK)
• Forceps
• Transfer pipettes
• Scale
• Magnetic stirrer
• Disposable pestle
• Glass storage vials
• Paraffin-embedding station
• Vacuum flask
• Filter
• Filter paper (Fisherbrand, cat no. 09-803-5C)
• Slides
• Coverslips
• Microtome
• Slide warmer
• Thermal cycler
• Nanodrop
• Gel box
• Water bath
• Slide mailers/coplin jars for dehydration and all other steps

Procedure

Generation of kidney-specific cDNA

1. Setup 1 male and 1 female mice for breeding. Check daily for plugs
2. 18 days from the day of plugging, prepare for embryo dissection: a. Rinse off forceps, transfer pipette, and dissection dishes with 70% ethanol. Fill dissection dishes with sterile PBS. b. Add ~400 uL of sterile PBS into an RNAse-free Eppendorf tube and place on ice.
3. Sacrifice pregnant mother and harvest E18.5 embryos. After removing each embryo from its amniotic sac, sacrifice embryos by decapitation. Carefully remove kidneys from embryos and remove any non-renal tissue from them (including the ureter, interstitial tissue, and adrenal glands). Transfer to Eppendorf tube containing PBS using the transfer pipette.
4. After harvesting all kidneys, remove as much PBS as possible from the tubes. Divide the kidneys into additional RNAse-free Eppendorf tubes so that each group of tissue weighs <30 mg (~5 kidneys).
5. Disrupt kidneys mechanically with disposable pestle and homogenize by adding 600 uL of Buffer RLT (from Qiagen RNeasy Mini Kit), transferring to QIAShredder columns, and centrifuging following manufacturer instructions.
6. Follow remaining Qiagen RNeasy Mini Kit instructions to extract RNA from the kidneys.
7. Quantify amount of RNA extracted with Nanodrop.
8. Generate cDNA with Superscript First-Strand Synthesis kit (Invitrogen) by following manufacturer’s protocol. a. Use between 1-2 ug of RNA per reaction. Multiple reactions may be necessary in order to use all extracted RNA. It is better to convert RNA into cDNA and store excess cDNA than store RNA as is. b. Use Oligo(dT) as the primers for the reaction.
9. Store cDNA at -20oC until ready to use.

Primer design and generation of PCR for template for probe synthesis

1. Design gene-specific primers for your gene(s) of interest using the Primer-BLAST tool available at http://www.ncbi.nlm.nih.gov/tools/primer-blast/. Importantly, change the minimum PCR product size to 500 and maximum to 2000 as products smaller than 500 or greater than 2000 do not work as well for downstream applications. a. Tip: If screening through multiple genes using this method, generate primers that will create products of varying sizes depending on the gene in order to barcode each gene. b. The software works best when given the Refseq mRNA record for the gene of interest.
2. After determining the 5’ and 3’ primers sequence, add the T7 promoter sequence (TAATACGACTCACTATAG) immediately 5’ to the 3’ primer. Therefore the sequence of the 3’ primer will be: 5’-TAATACGACTCACTATAGNNNNNNNNN-3’ where the Ns represent the sequence given for the 3’ primer from Primer-BLAST. The 5’ primer will remain unchanged. Order the primers through Integrated DNA Technologies
3. Once primers have arrived, resuspend primers in dH2O to make 100 mM solution, then mix and dilute 5’ and 3’ primers together by 5 to make a 20 mM working solution.
4. Prepare the following 25uL PCR reaction in 200 uL PCR tubes for each gene of interest:
   • 12.5 uL GoTaq
   • 1 uL cDNA
   • 2.5 uL 20mM primer mix
   • 9 uL dH2O
5. Run the PCR reactions on a thermal cycler using the following program:
   • Initial denaturation: 94.0oC 5:00 min
   • 45 cycles of:
   • Denaturation: 94.0oC 0:30 min
   • Annealing: 58.0oC 0:45 min
   • Extension: 72.0oC 3:00 min * Final extension: 72.0oC 5:00 min * End at 4oC
6. Once PCR reaction is complete, transfer reaction into 1.5 mL Eppendorf tube and bring reaction volume to 300 uL with dH2O.
7. Add 1 volume of Phenol:chloroform:isoamyl alcohol mixture to each reaction tube.
8. Vortex for 30 seconds, then immediately spin down at max speed for 5 minutes.
9. Transfer upper layer to a clean tube, and add 1 volume of chloroform to clean again. Vortex for 30 seconds, then spun down at max speed for 5 minutes.
10. Transfer upper layer to a clean tube. Be careful to not take any chloroform from the lower layer.
11. Add in 3M Na Acetate at 1/10 of total volume (app. 30 uL) to help precipitate DNA, 2uL of glycogen for visualization, and 3 volumes of -20oC cold 100% ethanol. Vortex to mix and incubate at -20oC for at least 30 minutes.
12. Centrifuge at max speed for 20 minutes and decant supernatant.
13. Washed with 500uL 70% ethanol and centrifuged at max speed for 5 minutes
14. Carefully removed as much ethanol as possible using a pipettor to avoid removing pellet. Let the DNA completely dry by leaving the tube open.
15. Resuspend in 30uL of dH2O.
16. Check DNA concentration and purity using Nanodrop.
17. To determine that the final clean PCR product is the correct size, mix 1 uL of DNA, 1uL of 6x loading dye, and 4 uL of dH2O together per sample and analyze the size of the product on a 1% agarose gel with gel electrophoresis.
18. Store cleaned PCR products at 4oC for short-term storage or -20oC for long-term storage

Probe Synthesis

1. Prepare the reagents provided in the T7 MEGAscript Kit (Ambion) by first creating 10 mM nucleotide solutions (from the provided stock of 75 mM) for each nucleotide by diluting in the provided H2O. Keep on ice. Warm up 10x reaction buffer solution until all of the spermidine dissolves into solution. Keep at RT.
2. Prepare the following 20 uL reactions in 1.5 mL Eppendorf tubes for each gene of interest at RT: a. 2 uL 10 mM ATP b. 2 uL 10 mM CTP c. 2 uL 10 mM GTP d. 1.33 uL 10 mM UTP e. 0.67 uL 10 mM Digoxigenin-11-UTP (Sigma) f. 2 uL 10x reaction buffer g. 2 uL T7 Enzyme mix (removed from -20oC only when needed)
3. Incubate reaction in 37oC water bath for >4 hours. The longer the reaction, the more product will be made. a. During this time, prepare a 1% agarose with enough lanes for 3x(number of samples) + appropriately sized ladder(s). b. Also warm up pre-hybridization buffer to 65oC.
4. After synthesis, remove 1 uL of sample and keep on ice for future gel.
5. Incubate samples with 1 uL DNAse (provided with MEGAscript kit) for 15 minutes at 37oC.
6. While samples are incubating with DNAse, prepare Micro Bio-Spin columns (BioRad) following manufacturer’s instructions.
7. Once DNAse digestion is complete, remove 1 uL from each sample and keep on ice for future gel. Apply the remainder of each sample onto the prepared Bio-spin columns and centrifuge following manufacturer’s instructions.
8. Once RNA probe has been cleaned, check the approximate concentration of each cleaned RNA probe sample using Nanodrop.
9. Additionally, remove 1 uL from each sample and keep on ice for gel. Prepare each sample at different stages of synthesis (after synthesis, after DNAse treatment, after cleaning with column) for gel electrophoresis by adding 1 uL of 6x loading dye and 4 uL of H2O to each sample.
10. Load 1% agarose gel so that the three samples per gene are arranged in order and run to fully separate bands.
11. While gel is running, add 500 uL of pre-hybridization buffer to each sample to stabilize RNA probe. Store at -20oC until needed.
12. Once gel has finished running, image to check for proper band sizes and concentration. a. Often, the T7 polymerase will make a probe size that is smaller than the original PCR product. Remember that sizes are approximate due to running RNA on an agarose gel and the addition of Digoxigenin within the RNA that will also affect how well it travels through the gel. b. Estimate concentration of the probe based off of the band intensity and Nanodrop reading, favoring the former. Band intensity equal to the fainter bands on DNA ladders are ~10x concentration. This value can be adjusted based off of the Nanodrop value given.

E15.5 tissue preparation and sectioning for slides

1. Setup 1 male and 1 female mice for breeding. Check daily for plugs.
2. 15 days from the day of plugging, sacrifice pregnant mother and harvest E15.5 embryos. After removing each embryo from its amniotic sac, sacrifice embryos by decapitation. In a dissection dish, carefully remove kidneys from embryos without damaging it. a. It is not necessary to remove surrounding tissues for these kidneys
3. Transfer to glass vial containing 4% PFA on ice. Fix overnight in 4% PFA at 4oC.
4. The next day, wash the kidneys with PBS 3x for 5 minutes each
5. Dehydrate the kidneys by washing them in a series of 25% ethanol, 50% ethanol, and 70% ethanol for 5 minutes each. After the last wash in 70% ethanol, kidneys can be stored at -20oC in 70% or 100% ethanol until needed.
6. When ready to embed tissues, transfer kidneys to 100% ethanol and wash for 5 minutes on belly dancer.
7. Wash in 100% ethanol for 30 minutes x2 on belly dancer
8. Wash in xylene for 10 minutes x2 on belly dancer
9. Remove xylene until a small amount remains on the tissue. At paraffin embedding station, add paraffin and wash for 10 minutes at 55oC
10. Wash in paraffin for 30 minutes at 55oC for at least 2 times a. Increasing the number of washes leads to improved tissue embedding and sectioning
11. Incubate kidneys overnight at 55oC a. Some paraffin embedding stations turn off overnight, so it may be necessary to transfer the tissues to an oven.
12. The next day, embed tissues in paraffin so that the kidney will be cut coronally (so that the entire renal structure can be seen on a single slice)
13. Once paraffin blocks have fully solidified, section kidneys with microtome in series of ~10-12 slides. Slides should be placed on slide warmer set at 42oC and have drops of dH2O on which to place kidney sections to help reduce tissue folding. Let slides dry overnight after the kidney is sectioned.
14. Once slides are completely dry, they are ready to be used immediately, or stored for future uses at 4oC.

In situ hybridization In situ hybridization protocol is a multi-day protocol and will be split up based on the days. Additionally, slides should be handled one at a time to prevent slides from drying out. Day 1:

1. Remove paraffin from slides by washing in xylene 2x for 10 minutes
2. Hydrate tissues by washing them in 100% ethanol 2x for 2 minutes, followed by 95%, 90%, 80%, 70%, 40% ethanol for 1 minute each.
3. Dip in H20, PBS 3x for 3 minutes.
4. Dilute proteinase K in PBS to get a working concentration of 15 ug/mL. Treat each slide with 500 uL of proteinase K for 15 minutes at room temperature. a. Timing is very important for this step. Treating slides with proteinase K for too little or too long may not lead to any noticeable expression or destroy the tissue. Carefully track the timing for each slide to ensure that each one has been incubated with proteinase K for 15 minutes.
5. Rinse in PBS 3x briefly.
6. Fix slides in 4% PFA for 5 minutes.
7. Rinse in PBS 3x for 3 minutes.
8. Warm pre-hybridization buffer to 65oC until the solution is completely clear. In a chamber humidified with 50% formamide/5x SSC, apply ~1ml hybridization buffer to each slide in a bolus. Incubate 45 – 60 minutes at RT.
9. While slides are blocking with pre-hybridization buffer, heat previously made RNA probes at 65oC for 5 minutes. Once RNA probes have been warmed so that the solution is clear, create 1x concentration dilutions of each probe and keep warmed at 65oC until needed.
10. After blocking, one slide at a time remove pre-hybridization buffer and apply 70-100 ul probe. Cover with glass coverslip. Incubate overnight at 65oC.
    a. Do this step quickly. As probe cools, it will precipitate.

Day 2:

1. Prewarm 5X SSC to 65oC and immerse slides for ~10 minutes at 65oC to allow coverslip to separate.
2. Prewarm 0.2X SSC to 65oC and wash slides in to 65oC 0.2X SSC 2x for 30 minutes.
3. While slides are washing in 0.2X SSC, Make 1ml/slide 2% blocking reagent (Roche) in 1x MBST. a. Warm the solution up to 65oC and mix frequently to get blocking reagent into solution
4. Rinse slides in 0.2X SSC at RT for 5 minutes. Transfer to 1x MBST
5. Working one at a time, arrange slides in chamber humidified with 1x MBST, remove excess MBST and apply 500 uL 2% blocking reagent in a bolus. Incubate at room temperature for at least 2 hours.
6. While slides are blocking, pre-block Anti-Digoxigenin-AP Fab fragments (1:4000, Roche) in remaining 2% blocking reagent in 4oC nutator for at least 1 hr.
7. After blocking, remove excess and apply 500 ul of Anti-Digoxigenin antibody to each slide. Cover with parafilm and incubate overnight at 4oC.

Day 3:

1. Immerse slides in 1x MBST to separate parafilm cover for ~5 minutes.
2. Rinse in 1x MBST with gentle agitation at room temperature 3x for 30 minutes.
3. While slides are washing in 1x MBST, prepare NTMT a. NTMT should always be made fresh the day it will be used.
4. Rinse slides in NTMT 3x for 5 minutes.
5. Prepare BM purple AP substrate, precipitating (Roche) by aliquoting the amount you need into a 50 mL conical and centrifuging the substrate at 1000x rpm for 1 minute to help remove any precipitate. Excess precipitate in the substrate may lead to dirtier staining on slides.
6. Immerse slides in BM purple substrate in a slide mailer. Develop at room temperature and monitor throughout the day. Reaction speed can greatly vary depending on the specific probe. Some probes may require multiple days of staining to see a positive signal. a. Development can be accelerated by incubating slides at 37oC instead of room temperature.
7. Once slides have adequately developed, fix slides in 4% PFA for at least 1-2 hours, preferably overnight.
8. Wash slides in PBS 3x for 5 minutes.

At this step, slides can be mounted as is and visualized, or stained with antibodies for immunofluorescence.

Mounting slides:

1. Dehydrate slides by washing them in 40%, 70%, 80%, 90%, and 95% ethanol for 1 minute each, followed by 100% ethanol wash 2x for 2 minutes
2. Wash slides in xylene 2x for 10 minutes
3. Mount slides with permount. Seal with nail polish.

Immunofluorescent co-stain: Note: not all fluorescent antibodies work with this method. Some optimization may be required depending on the antibody

1. Block slides with CAS-Block (Invitrogen) in a PBS-humidified chamber for at least 3 hours at room temperature.
2. While slides are blocking, pre-block primary antibody/antibodies of choice in CAS-block at appropriate concentration. For this work, we use Rat anti-PECAM and Rat anti-Endomucin antibodies at 1:100 concentration.
3. Once blocking is complete, incubate slides with primary antibody by adding ~100-200 uL of pre-blocked solution and covering with parafilm. Incubate overnight at 4oC.
4. The next day, wash slides in PBS 3x for 5 minutes.
5. Incubate slides with secondary antibody in CAS-Block for at least 1 hour at room temperature. For this work, we used Donkey anti-Rat 488 at 1:500 concentration.
6. Once secondary antibody incubation is complete, wash slides in PBS 3x for 5 minutes.
7. Mount with ProLong Gold antifade reagent (Life Technologies). Seal with nail polish.

Timing

cDNA and PCR generated probe synthesis, 1 day. In situ hybridization, 3 days.

Consortium

(Re)Building a Kidney (RBK) Consortium