. 2021 May 28;6:36. doi: 10.1038/s41525-021-00198-5

Table 3.

Summary of methods to measure telomere length.

Method	Principle	Analyte	Results	Strengths	Limitations
TRF^a^61,63	DNA fragments are visualised by Southern blot following digestion, and then compared against DNA ladder.	DNA (0.5–10 µg)	Average TL^k	▪ Reproducible ▪ Gives direct measurement of TL	▪ Large amount of DNA required. ▪ Subtelomeric polymorphisms impact data. ▪ Low hybridisation efficiency for very short telomeres. ▪ Risk of DNA degradation. ▪ Variation between labs (different restriction enzymes used).
qPCR^b mmqPCR^c aTLqPCR^d^61,64	qPCR: measures ratio between the telomere (T) and single-copy gene (S) amplification products mmqPCR: Telomere DNA and single-copy DNA amplified in the same tube. aTLqPCR: Standard curve from known TL	DNA (<100 ng)	T/S ratio^l (relative quantification)	qPCR: ▪ Small amount of DNA required ▪ High-throughput mmqPCR: ▪ Reduced human error related inaccuracy in qPCR aTLqPCR: ▪ Standard single-copy gene reference	▪ Large variation between different labs (different single-copy loci used). ▪ May not be of use in aneuploidy (single-copy gene duplicated or lost). ▪ Does not detect telomere-free ends ▪ Does not measure individual-specific lengths
STELA^e U-STELA^f ^61,68	STELA: Telomeric DNA amplified using subtelomeric- specific primers. U-STELA: DNA digestion followed by specific amplification for short telomeric fragments	Cells (1–1 × 10⁵⁾	Chromosome-specific TL	Both: ▪ Small amount of material required. ▪ Does not require viable cells STELA: ▪ Detects shortest telomeres on specific chromosomes. U-STELA: ▪ Detects shortest telomeres from every chromosome	Both: ▪ Unable to measure long TL. ▪ Sensitive to DNA input amount. ▪ Labour intensive. ▪ Low throughput. STELA: ▪ Requires highly specific subtelomeric sequences.
TeSLA^g⁶⁷	Southern blot with hypersensitive digoxigenin-labelled probes	DNA (<1 mcg)	Chromosome-specific TL	▪ Measures very short TL ▪ Detects longitudinal changes in TL	▪ Less suitable for cells with long heterogenous TL. ▪ Labour intensive. ▪ Low throughput.
Q-FISH^h (Interphase Q-FISH; Metaphase Q-FISH)^61,65,66	Telomere fluorescent intensity visualised after hybridisation with (CCCTAA)₃ probe.	Interphase Q-FISH: Interphase cells (can be done on fixed tissues and cells) Metaphase Q-FISH: Actively dividing cells (15–20 metaphase chromosomes)	Interphase Q-FISH: Average TL measured as relative fluorescence unit Metaphase Q-FISH: Average TL, chromosome-specific TL (both measured as relative fluorescence unit)	Interphase Q-FISH: ▪ Provides telomere length and histological information. ▪ Higher resolution. ▪ Less labour intensive than Metaphase Q-FISH. ▪ High-throughput Q-FISH available. ▪ Does not require mitotically active cells Metaphase Q-FISH: ▪ Suitable for telomeres of various lengths. ▪ Recognises “telomere-free” ends. ▪ Higher accuracy than Interphase Q-FISH.	Both ▪ Relative quantification. ▪ Labour intensive. Interphase Q-FISH: ▪ Unable to detect “telomere-free” ends. Metaphase Q-FISH: ▪ Requires mitotically active cells. ▪ Does not detect telomeres that are very short and do not hybridise with the probe. ▪ Skilled expertise needed for analysis
Flow FISH^61,66	Combination of flow cytometry with hybridisation of pantelomeric (CCCTAA)₃ probe to cells in suspension	White blood cells (0.5–2 × 10⁶)	Cell-specific average TL measured as relative fluorescent unit	▪ Cells can be sorted into subpopulations. ▪ Provides 3D telomeric signals within cells. ▪ May be adapted for a higher throughput.	▪ Sensitive to cell types (mostly done on peripheral blood mononuclear cells). ▪ Challenging to process suspension cells. ▪ Non-specific binding of telomeric probe. ▪ Does not detect chromosome-specific individual TL or telomere-free ends. ▪ Labour intensive. ▪ Costly.
PRINSⁱ⁶¹	Labels telomeric sequences in-situ on metaphase chromosomes/ interphase nuclei	15–20 metaphase chromosomes	Average TL and chromosome-specific TL (both are measured as relative fluorescence unit)	▪ Higher resolution ▪ Measures TL in specific cell types ▪ Can detect individual telomeres, telomere-free ends and average TL when used on metaphase chromosomes.	▪ Labour intensive. ▪ Relative quantification. ▪ Mitotically active cells required for metaphase chromosomes.
HPA^j⁶¹	Compares telomeric repeats and Alu repeats	10–3000 ng DNA	Average TL	▪ Quick ▪ Small amount of DNA required	▪ Measures mean telomere length only ▪ Alu repeats in sample can vary

Method

Principle

Analyte

Results

Strengths

Limitations

TRF^a^61,63

DNA fragments are visualised by Southern blot following digestion, and then compared against DNA ladder.

DNA

(0.5–10 µg)

Average TL^k

▪ Reproducible

▪ Gives direct measurement of TL

▪ Large amount of DNA required.

▪ Subtelomeric polymorphisms impact data.

▪ Low hybridisation efficiency for very short telomeres.

▪ Risk of DNA degradation.

▪ Variation between labs (different restriction enzymes used).

qPCR^b

mmqPCR^c

aTLqPCR^d^61,64

qPCR: measures ratio between the telomere (T) and single-copy gene (S) amplification products

mmqPCR: Telomere DNA and single-copy DNA amplified in the same tube.

aTLqPCR: Standard curve from known TL

DNA

(<100 ng)

T/S ratio^l (relative quantification)

qPCR:

▪ Small amount of DNA required

▪ High-throughput

mmqPCR:

▪ Reduced human error related inaccuracy in qPCR

aTLqPCR:

▪ Standard single-copy gene reference

▪ Large variation between different labs (different single-copy loci used).

▪ May not be of use in aneuploidy (single-copy gene duplicated or lost).

▪ Does not detect telomere-free ends

▪ Does not measure individual-specific lengths

STELA^e

U-STELA^f

^61,68

STELA: Telomeric DNA amplified using subtelomeric- specific primers.

U-STELA: DNA digestion followed by specific amplification for short telomeric fragments

Cells

(1–1 × 10⁵⁾

Chromosome-specific TL

Both:

▪ Small amount of material required.

▪ Does not require viable cells

STELA:

▪ Detects shortest telomeres on specific chromosomes.

U-STELA:

▪ Detects shortest telomeres from every chromosome

Both:

▪ Unable to measure long TL.

▪ Sensitive to DNA input amount.

▪ Labour intensive.

▪ Low throughput.

STELA:

▪ Requires highly specific subtelomeric sequences.

TeSLA^g⁶⁷

Southern blot with hypersensitive digoxigenin-labelled probes

DNA

(<1 mcg)

Chromosome-specific TL

▪ Measures very short TL

▪ Detects longitudinal changes in TL

▪ Less suitable for cells with long heterogenous TL.

▪ Labour intensive.

▪ Low throughput.

Q-FISH^h

(Interphase Q-FISH;

Metaphase Q-FISH)^61,65,66

Telomere fluorescent intensity visualised after hybridisation with (CCCTAA)₃ probe.

Interphase Q-FISH: Interphase cells (can be done on fixed tissues and cells)

Metaphase Q-FISH: Actively dividing cells (15–20 metaphase chromosomes)

Interphase Q-FISH:

Average TL measured as relative fluorescence unit

Metaphase Q-FISH: Average TL, chromosome-specific TL (both measured as relative fluorescence unit)

Interphase Q-FISH:

▪ Provides telomere length and histological information.

▪ Higher resolution.

▪ Less labour intensive than Metaphase Q-FISH.

▪ High-throughput Q-FISH available.

▪ Does not require mitotically active cells

Metaphase Q-FISH:

▪ Suitable for telomeres of various lengths.

▪ Recognises “telomere-free” ends.

▪ Higher accuracy than Interphase Q-FISH.

Both

▪ Relative quantification.

▪ Labour intensive.

Interphase Q-FISH:

▪ Unable to detect “telomere-free” ends.

Metaphase Q-FISH:

▪ Requires mitotically active cells.

▪ Does not detect telomeres that are very short and do not hybridise with the probe.

▪ Skilled expertise needed for analysis

Flow FISH^61,66

Combination of flow cytometry with hybridisation of pantelomeric (CCCTAA)₃ probe to cells in suspension

White blood cells

(0.5–2 × 10⁶)

Cell-specific average TL measured as relative fluorescent unit

▪ Cells can be sorted into subpopulations.

▪ Provides 3D telomeric signals within cells.

▪ May be adapted for a higher throughput.

▪ Sensitive to cell types (mostly done on peripheral blood mononuclear cells).

▪ Challenging to process suspension cells.

▪ Non-specific binding of telomeric probe.

▪ Does not detect chromosome-specific individual TL or telomere-free ends.

▪ Labour intensive.

▪ Costly.

PRINSⁱ⁶¹

Labels telomeric sequences in-situ on metaphase chromosomes/ interphase nuclei

15–20 metaphase chromosomes

Average TL and chromosome-specific TL (both are measured as relative fluorescence unit)

▪ Higher resolution

▪ Measures TL in specific cell types

▪ Can detect individual telomeres, telomere-free ends and average TL when used on metaphase chromosomes.

▪ Labour intensive.

▪ Relative quantification.

▪ Mitotically active cells required for metaphase chromosomes.

HPA^j⁶¹

Compares telomeric repeats and Alu repeats

10–3000 ng DNA

Average TL

▪ Quick

▪ Small amount of DNA required

▪ Measures mean telomere length only

▪ Alu repeats in sample can vary

References are in numerical superscript.

^aTerminal restriction fragment.

^bQuantitative polymerase chain reaction.

^c Monochrome multiplex polymerase chain reaction.

^dAbsolute telomere length quantitative polymerase chain reaction.

^eSingle-telomere length analysis.

^fUniversal single-telomere length analysis.

^gTelomere shortest length assay.

^hQuantitative fluorescence in-situ hybridisation.

ⁱPrimed in-situ subtype of Q-FISH.

^jHybridisation protection assay.

^kTelomere length.

^lTelomere repeat to single-copy gene ratio.