Total RNA was isolated from tissue, glands, 3D organoids, HGOs and 2D organoid monolayers using TRIzol (Life Technologies) according to manufacturer’s protocol. A High Capacity cDNA Reverse Transcription Kit synthesized cDNA from 100 ng of RNA following protocol provided by Applied Biosystems. Real-time PCR assays were utilized for the following genes in HGOs and 2D organoid monolayers: GAPDH (Hs02786624_g1), PD-L1 (Hs01125296_m1), SHH (Hs00179843_m1), TFF2 (Hs00193719_m1), Clustrin (Hs00156548_m1), and HE4 (Hs00899484_m1). Cell lineage markers were determined in tissue, glands, hFGOs, 2D monolayers fundic and antral HGOs by RT-PCR for Mucin 5AC (Hs01365616_m1), Mucin 6 (Hs01674026_g1), H+/K+ ATPase ATP4B (Hs01026288_m1), Pepsinogen C (Hs00160052_m1) Pepsinogen A (Hs05416800_g1) and Mist 1 (Hs00703572_s1). PCR amplifications were done with a pre-validated 20X TaqMan Expression Assay primers and a 2X TaqMan Universal Master Mix (Applied Biosystems) and a cDNA template in a total volume of 20 μL. Amplifications were performed in duplicate wells in a StepOne Real-Time PCR System (Applied Biosystems). Fold change was calculated at (Ct-Ct high) = n target, 2ntarget/2nGAPDEH = fold change where Ct = threshold cycle.
Taqman expression assay primers
Manufactured by Thermo Fisher Scientific
TaqMan Expression Assay primers are a set of oligonucleotides designed for the quantitative analysis of gene expression. The primers target specific gene sequences and are used in conjunction with TaqMan probes to measure mRNA levels in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Taqman universal master mix, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Allprep dna rna protein mini kit, by Qiagen (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (1 mentions)
4 protocols using taqman expression assay primers
1
Comprehensive Gene Expression Analysis in Organoids
2
Characterizing Gene Expression in Organoids
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Total RNA was isolated from mFGOs, hFGOs and gastric glands using TRIzol (Life
Technologies) according to manufacture’s protocol. A High Capacity cDNA
Reverse Transcription Kit synthesized cDNA from 100 ng of RNA following protocol
provided by Applied Biosystems. Real-time PCR assays were utilized for the following
genes in the mouse-derived organoids: GAPDH, alpha-Smooth Muscle Actin
(Mm00725412_s1), Zeb1 (Mm00495564_m1) and 2 (Mm0049713_m1), TWIST 1 (Mm04208233_g1)
and 2 (Mm000495564_m1), SNAIL 1 (Mm01249564_g1) and 2 (Mm00441531_m1). Cell lineage
markers were determined by RT-PCR for HK-ATPase (Hs01026288_m1), gastrin
(Hs01107047_m1), Muc5ac (Hs00873651_mH), and Muc6 (Hs01674026_g1). PCR amplifications
were done with pre-validated 20X TaqMan Expression Assay primers, 2X TaqMan Universal
Master Mix (Applied Biosystems), and cDNA template, in a total volume of 20
µL. Amplifications were performed with duplicate wells in a StepOne Real-Time
PCR System (Applied Biosytems), and fold change was calculated at (Ct-Ct high) = n
target, 2ntarget/2nGAPDH = fold change where Ct = threshold cycle.
Technologies) according to manufacture’s protocol. A High Capacity cDNA
Reverse Transcription Kit synthesized cDNA from 100 ng of RNA following protocol
provided by Applied Biosystems. Real-time PCR assays were utilized for the following
genes in the mouse-derived organoids: GAPDH, alpha-Smooth Muscle Actin
(Mm00725412_s1), Zeb1 (Mm00495564_m1) and 2 (Mm0049713_m1), TWIST 1 (Mm04208233_g1)
and 2 (Mm000495564_m1), SNAIL 1 (Mm01249564_g1) and 2 (Mm00441531_m1). Cell lineage
markers were determined by RT-PCR for HK-ATPase (Hs01026288_m1), gastrin
(Hs01107047_m1), Muc5ac (Hs00873651_mH), and Muc6 (Hs01674026_g1). PCR amplifications
were done with pre-validated 20X TaqMan Expression Assay primers, 2X TaqMan Universal
Master Mix (Applied Biosystems), and cDNA template, in a total volume of 20
µL. Amplifications were performed with duplicate wells in a StepOne Real-Time
PCR System (Applied Biosytems), and fold change was calculated at (Ct-Ct high) = n
target, 2ntarget/2nGAPDH = fold change where Ct = threshold cycle.
3
Quantitative RNA Expression Analysis in Fetal Tissues
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Total RNA was extracted from whole embryo (E9.5), yolk sac (E9.5 or E12.5), liver (E12.5) or placenta (E12.5) using an RNeasy Mini Kit (QIAGEN) or AllPrep DNA/RNA/Protein Mini Kit (QIAGEN). Total RNA treated with DNase I (QIAGEN) on-column was transcribed into first-strand cDNA using a SuperScript VILO Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. qRT-PCR analysis was performed by using TaqMan Expression Assay Primers (Applied Biosystems, Table S4). Expression levels were calculated by the relative standard curve method with 18S rRNA as an internal control and the data were shown as relative expression compared with the control group. In the comparison between fetal tissues and adult tissues, data were shown as fold increase over the average expression levels in the tissues of male C57BL/6J mice.
Table S4
4
Quantitative Analysis of Gene Expression in Mouse Stomach
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Total RNA was isolated from stomachs of uninfected and infected WT and PC-SHHKO mice. The High Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis from 100 ng of RNA following the recommended protocol (Applied Biosystems). Pre-designed real-time PCR assays were purchased for the following genes (Applied Biosystems): SHH (Mm00436528_m1), IFNγ (Mm01168134_m1), IL-1β (Mm01336189_m1) and mouse GAPDH (20X) (4352932-0803020), MIP2 (Mm00436450_m1). PCR amplifications were performed in a total volume of 20 µl, containing 20X TaqMan Expression Assay primers, 2X TaqMan Universal Master Mix (Applied Biosystems, TaqMan Gene Expression Systems) and cDNA template. Each PCR amplification was performed in duplicate wells in a StepOne Real-Time PCR System (Applied Biosystems), using the following conditions: 50°C 2 minutes, 95°C 10 minutes, 95°C 15 seconds (denature) and 60°C 1 minute (anneal/extend) for 40 cycles. IL-12 expression was quantified using specific primers as previously published for IL-12: Forward- 5′- GGA AGC ACG GCA GAA TA-3′ and Reverse- 5′- AAC TTG AGG GAG AAG TAG GAA TGG -3′ [70] (link) using the SYBR Green PCR Master Mix and protocol (Applied Biosystems). GAPDH was used as an internal control. See Methods S1 .
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