Hi,
I am interested in using q-RT-PCR to screen abnormal cellular processes such as ER stress, oxidative stress, autophagy, mitophagy, abnormal Golgi function, abnormal intracellular transport.
Can we list genes that can be used as indicative (maybe not always conclusive) expressional biomarkers (to detect with q-RT-PCR)?
If you have a successful, well-designed primers for SYBR green assay you may share it in here so they will be useful for others.
I have found following genes as markers of indicated processes. Do you have additional ones? Do you agree with these ones? Which ones are best according to your experience?
ER Stress: HERPUD1, EDEM1, GRP94, XBP1 (splicing not rt), BIP, CHOP
Autophagy: ATG12, ATG7, BECLIN1, VMP1, MAP1LC3, SQSTM1,
Oxidative Stress: GPX3,GPX4, GPX6, SOD2
Mitochondrial fusion/fission: FIS1, MFN1, MFN2
Mitochondrial integrity: Mithecondria specific (DNA)/Nuclear specific (DNA)
Golgi Stress: ????
Intracellular transport problems: ??
Others: ???
Thank you for your contributions.
Hi, I would like to suggest DNM1 as a marker for mitochondrial fusion/fission.
Regards, Christian
For oxidative stress, Nrf2, SODs, GPXs, NADPH oxidases, Catalase, TRX, GR may be measured at gene/protein levels. The problem is that many of these have several isoforms, then it depends on your tissue and overall signaling pathway you wanna dissect.
@Omer: DNM1L (regulator of mitochondrial and peroxisomal fission)
I am currently using following primers for indicated cellular stress pathways. But I did not test all of them with positive controls. If anybody would use these with positive controls, she can share her results here as a response for the benefit of scientific community.
ER Stress (q-RT-PCR):
CHOP_rtFrw GTCTAAGGCACTGAGCGTATCA
CHOP_rtRev CACTTCCTTCTTGAACACTCTCTC
BiP_Frw AACGCCAAGCAACCAAAGAC
Bip_Rev CTGCCGTAGGCTCGTTGAT
GRP94_Frw CGGTGTAGTGGGCGGA
GRP94_Rev GACCGACCCGAAGGTCA
EDEM1_Frw ACAACTACATGGCTCACGCC
EDEM1_Rev AGATTTGAAGGGTCCCCGC
HERPUD1_Frw AGCCTGCTGGTTCTAATCGG
HERPUD1_Rev GCTTCAGGCCTTGAGATGTTT
ER Stress (splicing, RT-PCR):
XBP1_Frw TTACGAGAGAAAACTCATGGCC
XBP1_Rev GGGTCCAAGTTGTCCAGAATGC Size of PCR products: unspliced XBP1 289 bp, spliced 263 bp.
Oxidative stress (I have not tried with a positive control)
GPX3_rtFrw AAGAGCTTGCACCATTCGGT
GPX3_rtRev CTGGTCGGACATACTTGAGGG
GPX6_rtFrw GGCTTGGCAGCTCAGTATCC
GPX6_rtRev ACCTGGACACACATACTTGAGAC
GPX4_rtFrw CAAGTACCGGGGCTTCGTG
GPX4_rtRev CCGAACTGGTTACACGGGAA
Mitochondrial DNA quantification:
MithSpcRtFrw CCAACAAACCTACCCACCCTT
MithSpcRtRev ATATTGATTTCACGGAGGATGGTG
HumDNASpcFrw GAGGAAGCACCAGCAGGATT
HumDNASpcRev TCTTCTCCAGCGCACCTTG
Mitochondrial abnormalities (Fusion and fission abnormalities)
MFN1rtFrw TCCGCCTTTAACTTCTCGGG
MFN1rtRev GCCATTATGCTAAGTCTCCGC
MFN2rtFrw ACCTCAACCTGAGACAGGACA
MFN2rtRev AAGAGCAGGGACATTGCGTTTTT
FIS1rtFrw CTTAAAGTACGTCCGCGGGT
FIS1rtRev GCCCACGAGTCCATCTTTCT
Autophagy:
VMP1rtFrv GGCCTGTGCTTCAATTCCAAAT
VMP1rtRev TGCTTTTCCAATTAGGGTTGCAC
BECN1rtFrw AAGTCGCTGAAGACAGAGCG
BECN1rtRev AGACCCTTCCATCCCTCAGC
MAP1LC3rtFrw GGCTATCGCCAGAGTCGGAT
MAP1LC3rtRev TCTTTGTTCGAAGGTGCGGC
p62SQSTM1Frw ATCTCCCGCCAGAGGCTG
p62SQSTM1Rev GCCGCTCCGATGTCATAGTT
ATG12rtFrw ACTCTCTATGAGTGTTTTGGCAGT
ATG12rtRev TCCATTTCATGTAGTAGCAAGTTGA
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