RNA in diagnostics
• 1.) direct RNA diagnostics – screening of whole coding region
of given gene
• 2.) gene – expression analysis:
– diferential diagnostics of some tumours
– detection of circulation tumour cells in blood and bone
marrow
– monitoring of course of therapy and detection of residual
disease
– control of graft before autologous transplatation
– differential display, PTT test, functional tests...
RNA in diagnostics
RNA
Mammalian cell:
• 10 - 30 pg total RNA
• rRNA (28S,18S, 5S) 80-85%
• tRNA, snRNA 15-20%
• mRNA 1-5%
360 000 mRNA molecules/cell,
12 000 different transcripts
typical length of 1 transcript cca 2kb
• presence of ribonucleases (RNases) in cell
• RNase
– very stable
– do not need cofactors
– efficient in low concentrations
– difficult inactivation
– contamination with RNases : human skin
dust particles (bacterias, fungi)
• isolation and analysis of RNA : special approach and
methods
RNA Unstability
•gene-expression analysis: analysed RNA must represent in vivo
expression of sample
•Complications - 1) reduction mRNA (downregulatoin of genes and
enzymatic degradation of RNA), 2) expression induction of certain
genes
RNA stabilisation in sample:
– immediately frost in liquid nitrogen and store in -80°C
– stabilisation solutions: RNAlater, PAXgene
Contamination with DNA
PCR primers overlapping border intron/exon
digestion with DNases
Stabilisation and storage of RNA
Direct RNA diagnostics
RNA A AA A A A A
T TT T TT T
T TT T TT TcDNA
PCR
RT-PCR
T TTT TT T primer oligo(dT)
gene specific primer
RNA Extraction
NF1 gene: 350 kb, 60 exons, 11 - 13 kb mRNA
- protein neurofibromin: 2818 aminoacids, probably
tumour supresor
RNA diagnostics of NF1 gene
Neurofibromatosis type 1
von Recklinhausen disease
Autosomal dominant
Frequency 1:3000
Locus 17q
50% mutations de novo
Predispositions to tumours of
neural system
Café-au- lait spots
Neurofibromas
Lisch nodule
Complications in molecular diagnostics of NF1
-problematic clinical diagnosis:
-up to 50 % cases de novo
-high mutation speed
-length of gene (350 kb, 60 exonů)
-absence of hot spots – need to search in whole gene
-unclear corelation between type of mutation and
manifestation
-different clinical manifestation even in patients with the
same mutation
Strategy of molecular – genetic testing of NF1
patients
NF1 DNA / RNA of pacient
Prenatal diagnostics
intragene DNA polymorfismus
IVS27AAAT2.1, IVS27AC28.4
IVS38GT53.0 - i 38
Linkage DNA analysis
DNA/RNA methods
DGGE, SSCP
cDNA / SSCP
Mutation analysis Direct sequencing
cDNA - SSCP analysis
RT
cDNA
PCR
NF1 cDNA ( 60 exons)
P1 P3 P5 P7 P9
P2 P4 P6 P8 P10
SSCP
Sequencing analysis
total RNA
Sequencing of cDNA segment P7 of NF1 gene ( exons 28 -32/33)
C >T
cDNA of NF1 pacient, mt C5839T ( Arg > STOP)
standard cDNA
5’ 3’
5’ 3’
Advantages and disadvantages of RNA
diagnostics
- Easier and faster screening
of multiexonic gene (10
segments of cDNA instead
of 60 exons), mRNA is
without introns
- Capture of splice mutations in
intrones
- Capture of deletion of whole
exonu on one allele
- cheaper
- More difficult taking blood for
RNA isolation
- Lower stability of RNA
- Longer segments – problems
with electroforetic separation
and sequencing
- Unclear effect of mutation on
phenotype level
Gene expression analysis
Real-time PCR method
 Real-Time PCR combines DNA amplification with real
time amplified product detection in a single tube
 Reliable
 Precise
 Fast
 Universal
 Variability of used probes
 Possibility of detection of more mutation during analysis
 Measurement of fluorescence
Determine differences in mRNA expression
 QUALITATIVE ANALYSIS
snp – single ncleotide polymorphism detection
 QUANTITATIVE ANALYSIS
amplicon amount detection
Detection methods
 Two types of Real-Time PCR detection chemistries:
 1. Specific Sequence Detection- Distinguishes between
a specific sequence of interest and non-specific
products. Can be used to detect different alleles
(TaqMan)
 2. Non-Specific Detection- Detects any dsDNA
produced during the reaction (SYBR green)
Tumour cells have different gene expression profile than healthy cells
Change in amount and spectrum of expressed mRNA
Relative quantification of expression relative to housekeeping gene
Housekeeping gene: control gene which is constant in the samples
Expression analysis in oncology
Oncomarkers
A tumor marker is a substance found in the blood, urine, or
body tissues that can be elevated in cancer. There are many
different tumor markers, each indicative of a particular disease
process, and they are used in oncology to help detect the
presence of cancer.
From substances produced by normal cells they differ
qualitatively – normal cells do not produce them, or
quantitatively – they are produced by both types of cells
• produced only in malignant cells
• organ specific
• in biological liquids in high concentrations
• level correlates with size of tumour, stage of disease, prognosis and
therapy effect
• provides evidence of residual tumour tissue
Ideal Oncomarker
Oncomarkes according to chemical structure or
biological function
• oncofetal antigens
• enzymes
• hormones
• intracellular oncomarkers
• other non specified substances
Onkomarkers: indication according to organ
 Prostate-Specific Antigen: PSA is prostate-specific, not
cancer-specific. A variety of conditions can raise PSA levels:
prostatitis (prostate inflammation), benign prostatic hypertrophy
(prostate enlargement), and prostate cancer. PSA levels can
also be influenced by a number of other things.
 Carcinoembryonic Antigen: Although CEA was first
indentified in colon cancer, an abnormal CEA blood level is
specific neither for colon cancer nor for malignancy in general.
Elevated CEA levels are found in a variety of cancers other
than colonic, including pancreatic, gastric, lung, and breast. It is
also detected in benign conditions including cirrhosis,
inflamatory bowel disease, chronic lung disease, and
pancreatitis. The CEA was found to be elevated in up to 19
percent of smokers and in 3 percent of a healthy control
population.
Thus, the test for oncomarker cannot substitute for a
pathological diagnosis.
Detection of minimal residual disease
Detection of presence of isolated tumour cells in blood, bone marrow
and lymphatic system – possible precursors of metastases
Imunohistochemistry – sensitivity 1 : 10 000
Flow cytometry – sensitivity 1 : 100 000
PCR – sensitivity 1: 1 000 000
Real- time PCR – sensitivity up 1 : 10 000 000