Aug 21, 2023
IgG sequencing of rat hybridoma
- 1Fred Hutching Cancer Center
Protocol Citation: Tamer B Shabaneh 2023. IgG sequencing of rat hybridoma. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9ppw1g3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2023
Last Modified: August 24, 2023
Protocol Integer ID: 86722
Abstract
The purpose of this protocol is to amplify IgG antibody variable regions derived from rat hybridoma RNA, using RT-PCR and Sanger sequencing.
Materials needed:
- RNA extraction: Qiagen RNEasy mini kit (74104)
- Reverse Transcription Kit: SMARTScribe Reverse Transcriptase (639537)
- DNA Polymerase: Invitrogen Platinum SuperFi II PCR Master Mix (12368010)
- Invitrogen's PureLink™ PCR Purification Kit #K310001
The layout of this protocol was adapted from a protocol composed by Andrew McGuire's laboratory at the Fred Hutchinson Cancer Center, which in turn was adapted from Meyer et al 2019 "A simplified workflow for monoclonal antibody sequencing." See attachment for the respective manuscript.
For this protocol, rat-specific primers were designed, as the immunization campaign was executed in rats. See attachment for the primer design strategy.
Primers
Primers
TS pF is ordered as RNA oligo; the remainder as DNA oligo
universal TS pF AAGCAGTGGTATCAACGCAGAGTACATrGrGrG
ISPCR pF aagcagtggtatcaacgcagag
rat IGHG RT pR GGACAGGGCTCCAGAGTTCC
rat IGHG PCR pR GACTGGCTCAGGGAAATAGCC
rat IGKC RT pR CTGATCAGTAACACTGTCCAGGAC
rat IGKC PCR pR CACTGATGTCTCTGGGATAGAAGTTG
rat IGLC1 RT pR GGGAGATAGGTGCACCATTTGC
rat IGLC1 PCR pR GGCCACTTCCACATCACTCG
rat IGLC2 RT pR TCCACACCCTGAGTGATAGGG
rat IGLC2 PCR pR CTTCCAGACCACTGTCATAACACC
Procedure
Procedure
RNA extraction: Extract RNA from the hybridoma sample using RNeasy total RNA kit, according to the manufacturer’s instructions.
Reverse Transcription: On ice, prepare the 1st reaction mix in PCR tubes for gamma chain and kappa chain (or lambda chain) cDNA synthesis according to the following recipe:
Gamma
| Component | Volume per rxn | |
| IGHG RT pR (10 uM) | 1 uL | |
| dNTP (10 mM) | 1 uL | |
| RNA sample (50 ng/uL) | 2 uL | |
| Total volume | 4 uL |
Kappa
| Component | Volume per rxn | |
| IGKC RT pR (10 uM) | 1 uL | |
| dNTP (10 mM) | 1 uL | |
| RNA sample (50 ng/uL) | 2 uL | |
| Total volume | 4 uL |
Lambda (include later if Kappa does not amplify)
| A | B | |
| Component | Volume per rxn | |
| IGLC1 (or C2) RT pR (10 uM) | 1 uL | |
| dNTP (10 mM) | 1 uL | |
| RNA sample (50 ng/uL) | 2 uL | |
| Total volume | 4 uL |
On ice, prepare a 2nd mastermix in Eppendorf tubes. This recipe if for one reaction regardless of chain type. Scale up for the number of samples as needed.
2nd reaction mix
| A | B | |
| Component | Volume per rxn | |
| 5x SMARTScribe buffer | 2 uL | |
| DTT (20 mM) | 1 uL | |
| Universal TS pR (100 uM) | 0.3 uL | |
| H2O | 1.70 uL | |
| Total volume | 5.00 uL |
After preparing the 2nd mastermix, incubate each of the 1st reaction mixes in a thermocycler for 3 minutes at 72°C.
While the incubation reaction is proceeding, add the following to the 2nd reaction mix:
| A | B | |
| Component | Vol. per rxn | |
| RNAse inhibitor (40 U/uL) | 0.50 uL | |
| SMARTScribe Rev. Transcriptase (100 U/uL) | 0.50 uL | |
| Total volume | 1.00 uL |
Once the incubation of the 1st reaction mix (from step 3.5) finishes, add 6 uL of the 2nd reaction mix to each tube of the 1st reaction mix.
With the 2 reaction mixes now combined, incubate each according to the following conditions:
| A | B | C | |
| Temperature | Time | Cycles | |
| 42°C | 60 min | 1 | |
| 70°C | 5 min | 1 | |
| 4°C | hold | - |
Proceed to PCR amplification step immediately after incubation has finished (once samples reach 4°C hold step).
PCR amplification: Prepare mastermix for PCR reaction according to following 2-step recipe:
Add the following components to each PCR tube.
Gamma
| A | B | |
| Component | Volume per rxn | |
| Platinum SuperFi II PCR MM | 25 uL | |
| ISPCR pF (10 uM) | 2.5 uL | |
| IGHG PCR pR (10 uM) | 2.5 uL | |
| cDNA from RT step (5-100ng) | 3 uL | |
| Water, nuclease-free | 17 uL | |
| Total volume | 50 uL |
Kappa
| A | B | |
| Component | Volume per rxn | |
| Platinum SuperFi II PCR MM | 25 uL | |
| ISPCR pF (10 uM) | 2.5 uL | |
| IGK PCR pR (10 uM) | 2.5 uL | |
| cDNA from RT step (5-100ng) | 3 uL | |
| Water, nuclease-free | 17 uL | |
| Total volume | 50 uL |
Lambda (if Kappa doesn’t amplify)
| A | B | |
| Component | Volume per rxn | |
| Platinum SuperFi II PCR MM | 25 uL | |
| ISPCR pF (10 uM) | 2.5 uL | |
| IGLC1 (or C2) PCR pR (10 uM) | 2.5 uL | |
| cDNA from RT step (5-100ng) | 3 uL | |
| Water, nuclease-free | 17 uL | |
| Total volume | 50 uL |
Cap each tube, then mix and briefly centrifuge the PCR tubes.
Place PCR tubes in thermocycler, and run according to the following conditions:
Gamma
| A | B | C | |
| Temperature | Time | Cycles | |
| 98°C | 30 sec | 1 | |
| 98°C | 15 sec | 35 | |
| 63°C | 30 sec | ||
| 72°C | 25 sec | ||
| 72°C | 5 min | 1 | |
| 4°C | hold | - |
Kappa/Lambda
| A | B | C | |
| Temperature | Time | Cycles | |
| 98°C | 30 sec | 1 | |
| 98°C | 15 sec | 35 | |
| 56°C | 30 sec | ||
| 72°C | 25 sec | ||
| 72°C | 5 min | 1 | |
| 4°C | hold | - |
Verify the gel bands
After PCR reaction completes, set up 1% agarose gel according to the following conditions:
In a flask, add the following components:
0.5 g – Agarose powder
50 mL – 1X TAE buffer
Put flask, with added reagents, in microwave and heat up until all the powder is fully dissolved (about 1 minute).
After microwaving is finished, remove flask with hot pad; add 5 uL of Sybrsafe to flask, swirl to mix. Pour into a tray with appropriate plastic comb, preferably one with wells that accommodate 20uL volumes.
Prepare a fraction of each PCR products to verify the band size on the gel. Aliquot 5 uL from each PCR tube into a new PCR strip, and add 1 uL of DNA Loading Dye to each sample.
Load the gel and run it for 25-30 minutes at 110 V. Modify these settings if needed.
Remove gel, image, and save an image copy for the records.
Note: There should be a gamma chain, and either a kappa or lambda chain.
Amplified rat antibody products: 550-600 bp.
A significant majority of mouse antibody light chains will be kappa; if kappa not present, repeat with IGLC1 and IGLC2 samples.
PCR cleanup
PCR samples that produce a band of the expected size can then be finalized using a PCR purification kit (e.g. Invitrogen's PureLink™ PCR Purification Kit #K310001), and eluted in 25 uL volume. Verify cDNA concentration by nanodrop.
Sanger sequencing should be performed (e.g. GeneWiz or Genomic Core).
Analyze the antibody nucleotide sequence
Open the website: https://www.imgt.org/IMGT_vquest/vquest
Copy and paste sequence into the IMGT webpage, in the section “sequence submission”.
Select “Rattus norvergicus” for species, and “IGH” as receptor type or locus.
Click on "Start".