Collagen-VI Specific Primer Design Identification in Rats
(Rattus norvegicus) Pancreas
Endin Nokik Stujanna1, Sri Suci Ningsih1, Rizkyana Avissa1, Novi Putri Ayu1,
Zahra Nurussofa1, Dewi Jantika Djuarna1, Rini Latifah1, Wawang Setiawan
Sukarya1
1

Faculty of Medicine,
Muhammadiyah Prof DR Hamka
University, Tangerang, Banten,
Indonesia
Correspondence:
Endin Nokik Stujanna,
Faculty of Medicine,
Muhammadiyah Prof DR Hamka
University, Raden Patah, Parung
Serab, Ciledug, Tangerang, Banten,
Indonesia
Zip Code: 13460
Email:
endin_stujanna@uhamka.ac.id
Received: 27 November, 2021
Revised: 15 January, 2022
Accepted: 22 February, 2022
Published: April 28, 2022
DOI: 10.33086/ijmlst.v4i1.2515

Abstract
Type 2 diabetes mellitus, the most common diabetes type
characterized by hyperglycemia, is caused by abnormal
secretion and activity of pancreatic insulin enzymes. The
extracellular matrix (ECM) plays a vital role in keeping β
pancreatic cells intact and undissociated. The ECM in the
pancreas can play a role in influencing insulin function and
production. The most abundant ECM in the pancreas is
collagen type VI. Collagen type VI has an essential role in
the survival of pancreatic islet cells, including pancreatic β
cells. Nowadays, Polymerase Chain Reaction (PCR)
technology is widely utilized for molecular biology analysis.
One of the most critical factors for successful Polymerase
Chain Reaction (PC)R is designing the correct specific PCR
primers. The objective of this study was to design a specific
primer for collagen VI in the pancreas of Rattus norvegicus.
The primer was designed and analyzed using MEGA.11,
primer three-plus, and primer-BLAST. Five primer pairs
were analyzed based on the characteristics of primer length,
product amplicon length, Tm value, GC percentage, and
secondary
structure.
Primer
pair
3
(F:5’TGTTTGGCTTTGTCGCGGGC-3’
and
R:5’TTGTTGCTGCCGACACTGGC-3’);
Col6a2
(F:5’TGTGGTCAACAGGCTGGGCG-3’
and
R:5’TCTGGCGCCGGCTCTCTTTG-3’) were considered as the
best primer for the Collagen VI expression detection from
the pancreas of Rattus norvegicus, which produce amplicon
about 250pb and 245pb, respectively.
Keywords
Diabetes Mellitus, Primer, Collagen VI, Rattus norvegicus.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author.

60

Endin Nokik Stujanna, et al.

of insulin secretion as the pancreatic islet of

INTRODUCTION
Type 2 diabetes mellitus (T2DM), a

Langerhans in vivo. The 3D culture model

global major health problem, ranks first as a

was considered more representative of

non-communicable disease that causes the

natural conditions in vivo (5).

highest death in Indonesia (1). DMT2 is

The synchronization between cells in the

closely related to the insulin hormone. This

pancreatic β islet will be weak if the cells

hormone is produced by pancreatic β cells,

dissociate. The extracellular matrix (ECM)

whose primary function for maintaining

plays a vital role in keeping pancreatic β cells

glucose homeostasis. Deficiency of insulin

intact and undissociated. ECM in the

secretion by pancreatic β cells is one of the

pancreas can play a role in protecting cells,

characteristics of the emergence of T2DM,

influencing insulin function and production,

which results in high blood glucose levels (2).

and

The glucose stimulates cellular signaling

pancreatic islets toward cytokines. ECM can

pathways to synthesize and translocate

be found in large quantities in the pancreas,

insulin granules. This series of processes is

including laminin, collagen type IV, and

supported by the pancreatic cell’s ability to

collagen type VI (6). The component of type

produce an oscillatory effect that triggers

VI collagen was higher than collagen types 1

insulin secretion. These oscillatory effects

and 4 in adult human pancreas research (7).

play a role in synchronizing secretory activity

Another

between pancreatic cells (3).

administration of type VI collagen in the

influencing

research

the

susceptibility

stated

that

of

the

The molecular and cellular mechanisms

pancreatic islets treatment could increase the

can be studied further by using an in vitro

viability of the pancreatic islet cells when

model using 3D spheroidal cultures of the

treated in vitro (8). It indicates that type VI

iGL cell line. iGL is a cell derived from a

collagen has an essential role in the survival

subculture of pancreatic β cells of rats

of pancreatic islet cells, including pancreatic

capable of expressing insulin-GLase in

cells. However, how type VI collagen

response to high environmental glucose

functions on pancreatic islet function and

levels (4). Research by Suzuki et al. (4) has

insulin expression is still unknown.
The role of type VI collagen was

spheroids exhibit an oscillatory effect of

explored by looking at the expression pattern

insulin secretion in response to high

of type VI collagen at the gene and protein

environmental glucose levels. Furthermore,

levels. Expression of type VI collagen at the

high glucose stimulation in the 3D culture of

gene level can be detected by the Polymerase

iGL cells showed a similar oscillatory effect

Chain Reaction (PCR) method. DNA primer

that

iGL

cell

Ina. J. Med. Lab. Sci. Tech. 2022; 4(1): 60–70

61

(insulin-GLase)

proved

Endin Nokik Stujanna, et al.

is DNA sequences complementary to the
sequence to be amplified. Therefore, the

This

research

used

bioinformatics

primer used in the PCR process serves as a

software online instruments for designing

barrier to the target DNA fragment to be

primer,

amplified. A pair of primers consists of a

https://www.bioinformatics.nl/cgi-

forward primer and a reverse primer (9).

bin/primer3plus/primer3plus.cgi), Integrated

The success of DNA amplification

62

MATERIALS AND METHODS

namely

Primer3Plus

(website:

DNA Technologies (IDT), BLAST (website:

depends on the accuracy of the primer. The

https://blast.ncbi.nlm.nih.gov/Blast.cgi)

primer

the National Center for Biotechnology

parameters

include

melting

at

temperature (Tm), percentage of G and C (%

Information

GC), 3′dimer, stability, repeats, and hairpins.

Evolutionary Genetic Analysis (MEGA). The

The primer designed must meet the criteria

material used in this research was DNA

used in the PCR process and produce

sequence data of collagen VI R. norvegicus

products according to the desired regional

downloaded in FASTA format from NCBI

range (10). This research was aimed to design

database. The method consists of searching

the primer with good specificity to detect the

gene sequences on the NCBI site in the gene

expression of the Cx36 gene (type VI

sub-search, selecting the “RefSeq” menu, and

collagen gene) in the pancreas of Rattus

selecting

norvegicus by PCR technique.

organism option (Figure 1).

(NCBI),

“Rattus

and

norvegicus”

Molecular

in

the

Figure 1. Collagen VI gene search result in NCBI
Ina. J. Med. Lab. Sci. Tech. 2022; 4(1): 60–70

Endin Nokik Stujanna, et al.

The gene sequences obtained may have

conserved sequences from these isoforms.

several isoforms. MEGA.11 application was

Then the conserved sequences were used for

used to carry out alignment to obtain

primer design with Primer3Plus (Figure 2).

Figure 2. Primer2Plus front page display

63

Ina. J. Med. Lab. Sci. Tech. 2022; 4(1): 60–70

Endin Nokik Stujanna, et al.

Figure 2 shows Primer2Plus setting.

length, Tm, %GC (11). In order to designing

Some settings on the “General and Advance

primers for PCR, two types of secondary

Settings” menu for the desired primer

structures should be analyzed, namely dimers

criteria. For example, it was related to the

and hairpins. The primers obtained were

primer

GC

further analyzed with IDT to determine how

composition, etc. Then select “Pick Primers”

big the primers were to form dimer and

to get the primer selection. The results

hairpin primer. Furthermore, the primers

displayed were several choices of primer

obtained were further analyzed with Primer-

pairs (forward and reverse) accompanied by

BLAST to determine the specificity of the

data on primer length, product or amplicon

primer to the target gene.

and

amplicon

size,

Tm,

Figure 3. Display of Primer-BLAST. results

64

RESULTS

DISCUSSION

The Collagen VI gene (col6a1: ID

The PCR method has been widely used

294337 and col6a2: ID 361821) in Rattus

in the biomolecular and medical world. Good

norvegicus is located on chromosome

primer design is an important factor for a

number 20 on NC_051355.1. This gene,

successful PCR process. Several essential

which has two isoforms, is aligned using the

characteristics that need to be considered in

MEGA application. The primer design result

designing PCR were Primer length, GC

was carried out by using Primer3Plus and

composition, melting temperature (Tm),

further analyzed with IDT and Primer-

dimer primer, and hairpin primer (12). The

BLAST, which are summarised in the

recommended primer length for optimum

following Tabel 1.

PCR application is 18-30 base length (bp). If
Ina. J. Med. Lab. Sci. Tech. 2022; 4(1): 60–70

Endin Nokik Stujanna, et al.

the primer is too short, it will cause non-

the recommended range of 58.8-59.6°C.

specific amplification, while too long primer

Only col6a1 1 (forward, reverse), 2 (reverse),

tends to form secondary structures such as

3 (reverse), and 4 (forward) primer pairs have

hairpin loops (13). All primers designed for

a Tm value slightly higher than the

Collagen VI had a length of 20 bp, which was

recommended value of 60.2°C, and only

categorized as a good primer.

col6a2 primer pairs 2 (reverse), which had a

The GC composition in the primer was
also essential to note. The GC proportion was

Tm

value

slightly

higher

than

the

recommended value of 60.0°C.

the percentage that described the ratio of G

An important factor to be considered in

and C nucleotides presented in the primer

designing a good primer was the possibility

sequence. The primer GC proportion was 30-

of primer forming a secondary structure. The

80%. However, the recommended optimal

secondary structure can be formed from one

GC proportion was at a value of 40-60% (12).

primer sequence itself that forms a hairpin

The primer design results obtained had a

loop structure or between pairs of primer that

value of 60-65%. Although this design value

form dimer primer or heterodimer (12,13).

was

GC

This secondary structure should be avoided

composition was considered less adverse on

because it can reduce the specificity of the

the PCR results (10). The nucleotide

primer.

slightly

higher,

the

higher

composition was also closely related to the
melting temperature (Tm) value.

All primers could form self-dimer or
heterodimer structures based on the primer

The reaction specificity of the PCR was

design results. The primer pair with the

highly dependent on the primer Tm value.

lowest selfdimer, heterodimer, and hairpin

The recommended optimal Tm value was in

numbers was the number four primer pair

the range of 50-60°C. The two primers

with selfdimer and hairpin values 1-2 and

should have the same Tm value or only had a

heterodimer 15.

difference of about 2-3°C. Different Tm can

The five pairs of primer obtained were

cause a decrease in the primer annealing

further analyzed for specificity using Primer-

efficiency (13).

BLAST (11). All tested primer pairs showed

All primer pairs obtained from the

specific results with the Col6a1 gene in

Collagen VI primer design had a Tm value

Rattus norvegicus (>XM_215375.8 Rattus

difference of not more than 3°C between the

norvegicus collagen type VI alpha 1 chain

forward and reversed primer. In addition,

(Col6a1), mRNA) with a product amplicon

almost all primer pairs had Tm values within

length of 247pb.

65

Ina. J. Med. Lab. Sci. Tech. 2022; 4(1): 60–70

Endin Nokik Stujanna, et al.

Table 1. Collagen VI primer design results
Gene
Name
Col6a
1

66

Col6a
2

primary
length
(bp)

product
amplico
n length
(bp)
F-20
R-20

Tm
(°C)

GC
(%)

Selfdimer

Hetero
dimer

F-60,2
R-60,2

F-65
R-65

F-10
R-12

Gene ID

Primer sequences

>lcl|NC_05
1355.1_cds
_XP_2153
75.5_1

F-TTGGCTTTGTCGCGGGCTCC
R-TTGTGGCTGCCGACACTGGC

247

F-TGACCGGCTGAGCAAGGACG
R-ATGTGCCGCCAGCCATCCAC

179

F-20
R-20

F-59,1
R-60,0

F-65
R-65

F- TGTTTGGCTTTGTCGCGGGC
R- TTGTGGCTGCCGACACTGGC

250

F-20
R-20

F-59,0
R-60,2

F- TGGCTGGCGGCACATTCACC
R- ACGTTGAGCTGGTCGGAGCC

223

F-20
R-20

F- TGACCGGCTGAGCAAGGACG
R- TCCGGTGAATGTGCCGCCAG

187

F-TGAGCTGGCGCTATGGTGGC
R-ACGTGCTGCCGGATCTGCTG

172

>lcl|NC_05
1355.1_cds
_NP_0010
94211.1_1

Hairpin

BLAST result

13

F-4
R-3

F-8
R-12

17

F-4
R-1

F-65
R-65

F-8
R-9

13

F-5
R-3

F-60,2
R-59,1

F-60
R-65

F-13
R-10

15

F-1
R-2

F-20
R-20

F-59,1
R-59,7

F-65
R-65

F-8
R-10

16

F-4
R-2

F-20
R-20

F-59,5
R-59,8

F-65
R-65

F-12
R-10

15

F-2
R-4

>XM_215375.8 PREDICTE
D: Rattus norvegicus
collagen type VI alpha 1
chain (Col6a1), mRNA.
247bp.
>XM_215375.8 PREDICTE
D: Rattus norvegicus
collagen type VI alpha 1
chain (Col6a1), mRNA.
179bp.
>XM_215375.8 PREDICTE
D: Rattus norvegicus
collagen type VI alpha 1
chain (Col6a1), mRNA.
250bp.
>XM_215375.8 PREDICTE
D: Rattus norvegicus
collagen type VI alpha 1
chain (Col6a1), mRNA.
223bp.
>XM_215375.8 PREDICTE
D: Rattus norvegicus
collagen type VI alpha 1
chain (Col6a1), mRNA.
187bp.
>XM_006256300.4 PREDI
CTED: Rattus norvegicus
collagen type VI alpha 2
chain (Col6a2), transcript
variant X1, mRNA.
172bp.

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Endin Nokik Stujanna, et al.
Gene
Name

Gene ID

Primer sequences

primary
length
(bp)

product
amplico
n length
(bp)
F-20
R-20

Tm
(°C)

GC
(%)

Selfdimer

Hetero
dimer

Hairpin

BLAST result

>lcl|NC_05
1355.1_cds
_XP_0062
56362.1_2

F-TTCCGCAGGGGCACCTTCAC
R-ACGGCAAAGAGCCGGATGCC

188

F-59,3
R-60,0

F-65
R-65

F-9
R-9

19

F-4
R-1

245

F-20
R-20

F-59,6
R-59,4

F-65
R-65

F-11
R-7

13

F-5
R-1

F- TTCCGCAGGGGCACCTTCAC
R- TTGGGGGCCACGGCAAAGAG

197

F-20
R-20

F-59,3
R-59,5

F-65
R-65

F-9
R-9

16

F-4
R-1

F- ATGCCCAGCAGCAGGAAGCC
R- TAGGCCACCATAGCGCCAGC

248

F-20
R-20

F-59,7
R-58,9

F-65
R-65

F-14
R-15

18

F-5
R-1

>XM_006256300.4 PREDI
CTED: Rattus norvegicus
collagen type VI alpha 2
chain (Col6a2), transcript
variant X1, mRNA.
188bp.
>XM_006256300.4 PREDI
CTED: Rattus norvegicus
collagen type VI alpha 2
chain (Col6a2), transcript
variant X1, mRNA.
245bp.
>XM_006256300.4 PREDI
CTED: Rattus norvegicus
collagen type VI alpha 2
chain (Col6a2), transcript
variant X1, mRNA.
197bp.
>XM_006256300.4 PREDI
CTED: Rattus norvegicus
collagen type VI alpha 2
chain (Col6a2), transcript
variant X1, mRNA.
248bp.

F- TGTGGTCAACAGGCTGGGCG
R- TCTGGCGCCGGCTCTCTTTG

67

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Endin Nokik Stujanna, et al.

68

Figure 4. Position of primers in the gene col6a2

Thus, primer pair 3 was considered the

selection of designed primer outcome must

closest to a good primer category for

be considered after performing experimental

detecting Collagen VI expression from the

tests in the laboratory.

pancreas of Rattus norvegicus at the gene

Researchers can redesign the primer if

level by PCR method with amplicon length.

the PCR results are not good by changing the

250pb product. As for the Col6a2 gene in

primary parameter criteria settings. For

Rattus

(>XM_006256300.4

example, change the GC proportion value,

Rattus norvegicus collagen type VI alpha 2

shift the Tm number, increase the resulting

chain (Col6a2), mRNA) with a product

amplicon length to 150-259pb [12], or use

amplicon length of 245pb.

other primary design applications available

norvegicus

Besides all the considerations above, the

online for free.

Ina. J. Med. Lab. Sci. Tech. 2022; 4(1): 60–70

Endin Nokik Stujanna, et al.

contributions to conception and design,

CONCLUSIONS
The

applications

MEGA.11,

acquisition

of

data,

or

analysis

and

can

interpretation of data. Rizkyana Avissa:

facilitate designing the specific Collagen VI

Substantial contributions to conception and

primers. Col6a1 gene primer pairs 3 (F:5’-

design, acquisition of data, or analysis and

TGTTTGGCTTTGTCGCGGGC-3’

and

interpretation of data. Novi Putri Ayu:

R:5’-TTGTTGCTGCCGACACTGGC-3’);

Substantial contributions to acquisition of

Col6a2

data.

Primer3Plus,

and

Primer-BLAST

(F:5’-

TGTGGTCAACAGGCTGGGCG-3’

and

Zahra

Nurussofa:

Substantial

contributions to conception and design

R:5’-TCTGGCGCCGGCTCTCTTTG-3’)

Dewi

Jantika

Djuarna:

Substantial

are the best primer in this study for Collagen

contributions to conception and design

VI expression detection in Rattus norvegicus

Rini Latifah: Substantial contributions to

pancreas and produce amplicon around

conception and design. Wawang Setiawan

250pb and 245pb, respectively. Future

Sukarya: Final approval of the version to be

research is required to analyze the specificity

published

of the primer design.

ACKNOWLEDGMENTS
AUTHOR CONTRIBUTIONS
Endin

Nokik

Stujanna:

The authors would like to thank all

Substantial

contributions to conception and design,
acquisition

of

data,

or

analysis

supporting staff at the Faculty of Medicine
and thank Lemlit UHAMKA.

and

interpretation of data, Drafting the article or

CONFLICT OF INTEREST

revising it critically for important intellectual
content. Sri Suciati Ningsih: Substantial

The authors declare that there is no
conflict of interest.

2020:21(5).
https://doi.org/10.3390/ijms21051770.

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