60 

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  
 

 
1Faculty 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. 

 

 

 



 

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INTRODUCTION 

Type 2 diabetes mellitus (T2DM), a 

global major health problem, ranks first as a 

non-communicable disease that causes the 

highest death in Indonesia (1). DMT2 is 

closely related to the insulin hormone. This 

hormone is produced by pancreatic β cells, 

whose primary function for maintaining 

glucose homeostasis. Deficiency of insulin 

secretion by pancreatic β cells is one of the 

characteristics of the emergence of T2DM, 

which results in high blood glucose levels (2). 

The glucose stimulates cellular signaling 

pathways to synthesize and translocate 

insulin granules. This series of processes is 

supported by the pancreatic cell’s ability to 

produce an oscillatory effect that triggers 

insulin secretion. These oscillatory effects 

play a role in synchronizing secretory activity 

between pancreatic cells (3). 

The molecular and cellular mechanisms 

can be studied further by using an in vitro 

model using 3D spheroidal cultures of the 

iGL cell line. iGL is a cell derived from a 

subculture of pancreatic β cells of rats 

capable of expressing insulin-GLase in 

response to high environmental glucose 

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

proved that iGL cell (insulin-GLase) 

spheroids exhibit an oscillatory effect of 

insulin secretion in response to high 

environmental glucose levels. Furthermore, 

high glucose stimulation in the 3D culture of 

iGL cells showed a similar oscillatory effect 

of insulin secretion as the pancreatic islet of 

Langerhans in vivo. The 3D culture model 

was considered more representative of 

natural conditions in vivo (5). 

The synchronization between cells in the 

pancreatic β islet will be weak if the cells 

dissociate. The extracellular matrix (ECM) 

plays a vital role in keeping pancreatic β cells 

intact and undissociated. ECM in the 

pancreas can play a role in protecting cells, 

influencing insulin function and production, 

and influencing the susceptibility of 

pancreatic islets toward cytokines. ECM can 

be found in large quantities in the pancreas, 

including laminin, collagen type IV, and 

collagen type VI (6). The component of type 

VI collagen was higher than collagen types 1 

and 4 in adult human pancreas research (7). 

Another research stated that the 

administration of type VI collagen in the 

pancreatic islets treatment could increase the 

viability of the pancreatic islet cells when 

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

collagen has an essential role in the survival 

of pancreatic islet cells, including pancreatic 

cells. However, how type VI collagen 

functions on pancreatic islet function and 

insulin expression is still unknown.   

The role of type VI collagen was 

explored by looking at the expression pattern 

of type VI collagen at the gene and protein 

levels. Expression of type VI collagen at the 

gene level can be detected by the Polymerase 

Chain Reaction (PCR) method. DNA primer 



 

 

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is DNA sequences complementary to the 

sequence to be amplified. Therefore, the 

primer used in the PCR process serves as a 

barrier to the target DNA fragment to be 

amplified. A pair of primers consists of a 

forward primer and a reverse primer (9). 

The success of DNA amplification 

depends on the accuracy of the primer. The 

primer parameters include melting 

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

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

The primer designed must meet the criteria 

used in the PCR process and produce 

products according to the desired regional 

range (10). This research was aimed to design 

the primer with good specificity to detect the 

expression of the Cx36 gene (type VI 

collagen gene) in the pancreas of Rattus 

norvegicus by PCR technique. 

 

MATERIALS AND METHODS  

This research used bioinformatics 

software online instruments for designing 

primer, namely Primer3Plus (website: 

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

bin/primer3plus/primer3plus.cgi), Integrated 

DNA Technologies (IDT), BLAST (website: 

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

the National Center for Biotechnology 

Information (NCBI), and Molecular 

Evolutionary Genetic Analysis (MEGA). The 

material used in this research was DNA 

sequence data of collagen VI R. norvegicus 

downloaded in FASTA format from NCBI 

database. The method consists of searching 

gene sequences on the NCBI site in the gene 

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

selecting “Rattus norvegicus” in the 

organism option (Figure 1).

 

Figure 1. Collagen VI gene search result in NCBI 



 

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The gene sequences obtained may have 

several isoforms. MEGA.11 application was 

used to carry out alignment to obtain 

conserved sequences from these isoforms. 

Then the conserved sequences were used for 

primer design with Primer3Plus (Figure 2).  

 

 

 
Figure 2. Primer2Plus front page display 

 



 

 

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Figure 2 shows Primer2Plus setting. 

Some settings on the “General and Advance 

Settings” menu for the desired primer 

criteria. For example, it was related to the 

primer and amplicon size, Tm, GC 

composition, etc. Then select “Pick Primers” 

to get the primer selection. The results 

displayed were several choices of primer 

pairs (forward and reverse) accompanied by 

data on primer length, product or amplicon 

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

primers for PCR, two types of secondary 

structures should be analyzed, namely dimers 

and hairpins. The primers obtained were 

further analyzed with IDT to determine how 

big the primers were to form dimer and 

hairpin primer. Furthermore, the primers 

obtained were further analyzed with Primer-

BLAST to determine the specificity of the 

primer to the target gene. 

 

 
Figure 3. Display of Primer-BLAST. results 

 

RESULTS 

The Collagen VI gene (col6a1: ID 

294337 and col6a2: ID 361821) in Rattus 

norvegicus is located on chromosome 

number 20 on NC_051355.1. This gene, 

which has two isoforms, is aligned using the 

MEGA application. The primer design result 

was carried out by using Primer3Plus and 

further analyzed with IDT and Primer-

BLAST, which are summarised in the 

following Tabel 1. 

DISCUSSION 

The PCR method has been widely used 

in the biomolecular and medical world. Good 

primer design is an important factor for a 

successful PCR process. Several essential 

characteristics that need to be considered in 

designing PCR were Primer length, GC 

composition, melting temperature (Tm), 

dimer primer, and hairpin primer (12). The 

recommended primer length for optimum 

PCR application is 18-30 base length (bp). If 



 

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the primer is too short, it will cause non-

specific amplification, while too long primer 

tends to form secondary structures such as 

hairpin loops (13). All primers designed for 

Collagen VI had a length of 20 bp, which was 

categorized as a good primer. 

The GC composition in the primer was 

also essential to note. The GC proportion was 

the percentage that described the ratio of G 

and C nucleotides presented in the primer 

sequence. The primer GC proportion was 30-

80%. However, the recommended optimal 

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

The primer design results obtained had a 

value of 60-65%. Although this design value 

was slightly higher, the higher GC 

composition was considered less adverse on 

the PCR results (10). The nucleotide 

composition was also closely related to the 

melting temperature (Tm) value. 

The reaction specificity of the PCR was 

highly dependent on the primer Tm value. 

The recommended optimal Tm value was in 

the range of 50-60°C. The two primers 

should have the same Tm value or only had a 

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

cause a decrease in the primer annealing 

efficiency (13).  

All primer pairs obtained from the 

Collagen VI primer design had a Tm value 

difference of not more than 3°C between the 

forward and reversed primer. In addition, 

almost all primer pairs had Tm values within 

the recommended range of 58.8-59.6°C. 

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

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

a Tm value slightly higher than the 

recommended value of 60.2°C, and only 

col6a2 primer pairs 2 (reverse), which had a 

Tm value slightly higher than the 

recommended value of 60.0°C.  

An important factor to be considered in 

designing a good primer was the possibility 

of primer forming a secondary structure. The 

secondary structure can be formed from one 

primer sequence itself that forms a hairpin 

loop structure or between pairs of primer that 

form dimer primer or heterodimer (12,13). 

This secondary structure should be avoided 

because it can reduce the specificity of the 

primer.  

All primers could form self-dimer or 

heterodimer structures based on the primer 

design results. The primer pair with the 

lowest selfdimer, heterodimer, and hairpin 

numbers was the number four primer pair 

with selfdimer and hairpin values 1-2 and 

heterodimer 15.  

The five pairs of primer obtained were 

further analyzed for specificity using Primer-

BLAST (11). All tested primer pairs showed 

specific results with the Col6a1 gene in 

Rattus norvegicus (>XM_215375.8 Rattus 

norvegicus collagen type VI alpha 1 chain 

(Col6a1), mRNA) with a product amplicon 

length of 247pb. 

 



 

 

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Table 1. Collagen VI primer design results 

Gene 

Name 
Gene ID Primer sequences 

primary 

length 

(bp) 

product 

amplico

n length 

(bp) 

Tm 

(°C) 

GC 

(%) 

Self-

dimer 

Hetero 

dimer 
Hairpin BLAST result 

Col6a

1 

>lcl|NC_05

1355.1_cds

_XP_2153

75.5_1 

F-TTGGCTTTGTCGCGGGCTCC 

R-TTGTGGCTGCCGACACTGGC 

247 F-20 

R-20 

F-60,2 

R-60,2 

F-65 

R-65 

F-10 

R-12 

13 F-4 

R-3 

>XM_215375.8 PREDICTE

D: Rattus norvegicus 

collagen type VI alpha 1 

chain (Col6a1), mRNA. 

247bp. 

  F-TGACCGGCTGAGCAAGGACG 

R-ATGTGCCGCCAGCCATCCAC 

179 F-20 

R-20 

F-59,1 

R-60,0 

F-65 

R-65 

F-8 

R-12 

17 F-4 

R-1 

>XM_215375.8 PREDICTE

D: Rattus norvegicus 

collagen type VI alpha 1 

chain (Col6a1), mRNA. 

179bp. 

  F- TGTTTGGCTTTGTCGCGGGC 

R- TTGTGGCTGCCGACACTGGC 

250 F-20 

R-20 

F-59,0 

R-60,2 

F-65 

R-65 

F-8 

R-9 

13 F-5 

R-3 

>XM_215375.8 PREDICTE

D: Rattus norvegicus 

collagen type VI alpha 1 

chain (Col6a1), mRNA. 

250bp. 

  F- TGGCTGGCGGCACATTCACC 

R- ACGTTGAGCTGGTCGGAGCC 

223 F-20 

R-20 

F-60,2 

R-59,1 

F-60 

R-65 

F-13 

R-10 

15 F-1 

R-2 

>XM_215375.8 PREDICTE

D: Rattus norvegicus 

collagen type VI alpha 1 

chain (Col6a1), mRNA. 

223bp. 

  F- TGACCGGCTGAGCAAGGACG 

R- TCCGGTGAATGTGCCGCCAG 

187 F-20 

R-20 

F-59,1 

R-59,7 

F-65 

R-65 

F-8 

R-10 

16 F-4 

R-2 

>XM_215375.8 PREDICTE

D: Rattus norvegicus 

collagen type VI alpha 1 

chain (Col6a1), mRNA. 

187bp. 

Col6a

2 

>lcl|NC_05

1355.1_cds

_NP_0010

94211.1_1 

F-TGAGCTGGCGCTATGGTGGC 

R-ACGTGCTGCCGGATCTGCTG 

172 F-20 

R-20 

F-59,5 

R-59,8 

F-65 

R-65 

F-12 

R-10 

15 F-2 

R-4 

>XM_006256300.4 PREDI

CTED: Rattus norvegicus 

collagen type VI alpha 2 

chain (Col6a2), transcript 

variant X1, mRNA. 

172bp. 

 

https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483


 

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Gene 

Name 

Gene ID Primer sequences primary 

length 

(bp) 

product 

amplico

n length 

(bp) 

Tm 

(°C) 

GC 

(%) 

Self-

dimer 

Hetero 

dimer 

Hairpin BLAST result 

 >lcl|NC_05

1355.1_cds

_XP_0062

56362.1_2 

F-TTCCGCAGGGGCACCTTCAC 

R-ACGGCAAAGAGCCGGATGCC 

188 F-20 

R-20 

F-59,3 

R-60,0 

F-65 

R-65 

F-9 

R-9 

19 F-4 

R-1 

>XM_006256300.4 PREDI

CTED: Rattus norvegicus 

collagen type VI alpha 2 

chain (Col6a2), transcript 

variant X1, mRNA. 

188bp. 

  F- TGTGGTCAACAGGCTGGGCG 

R- TCTGGCGCCGGCTCTCTTTG 

245 F-20 

R-20 

F-59,6 

R-59,4 

F-65 

R-65 

F-11 

R-7 

13 F-5 

R-1 

>XM_006256300.4 PREDI

CTED: Rattus norvegicus 

collagen type VI alpha 2 

chain (Col6a2), transcript 

variant X1, mRNA. 

245bp. 

  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 

>XM_006256300.4 PREDI

CTED: Rattus norvegicus 

collagen type VI alpha 2 

chain (Col6a2), transcript 

variant X1, mRNA. 

197bp. 

  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. 

248bp. 

 

https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483
https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483


 

 

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Figure 4. Position of primers in the gene col6a2 

 

Thus, primer pair 3 was considered the 

closest to a good primer category for 

detecting Collagen VI expression from the 

pancreas of Rattus norvegicus at the gene 

level by PCR method with amplicon length. 

250pb product. As for the Col6a2 gene in 

Rattus norvegicus (>XM_006256300.4 

Rattus norvegicus collagen type VI alpha 2 

chain (Col6a2), mRNA) with a product 

amplicon length of 245pb. 

Besides all the considerations above, the 

selection of designed primer outcome must 

be considered after performing experimental 

tests in the laboratory.  

Researchers can redesign the primer if 

the PCR results are not good by changing the 

primary parameter criteria settings. For 

example, change the GC proportion value, 

shift the Tm number, increase the resulting 

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

other primary design applications available 

online for free. 



 

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CONCLUSIONS 

The applications MEGA.11, 

Primer3Plus, and Primer-BLAST can 

facilitate designing the specific Collagen VI 

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

TGTTTGGCTTTGTCGCGGGC-3’ and 

R:5’-TTGTTGCTGCCGACACTGGC-3’); 

Col6a2 (F:5’-

TGTGGTCAACAGGCTGGGCG-3’ and 

R:5’-TCTGGCGCCGGCTCTCTTTG-3’) 

are the best primer in this study for Collagen 

VI expression detection in Rattus norvegicus 

pancreas and produce amplicon around 

250pb and 245pb, respectively. Future 

research is required to analyze the specificity 

of the primer design. 

 

AUTHOR CONTRIBUTIONS  

Endin Nokik Stujanna: Substantial 

contributions to conception and design, 

acquisition of data, or analysis and 

interpretation of data, Drafting the article or 

revising it critically for important intellectual 

content. Sri Suciati Ningsih: Substantial 

contributions to conception and design, 

acquisition of data, or analysis and 

interpretation of data. Rizkyana Avissa: 

Substantial contributions to conception and 

design, acquisition of data, or analysis and 

interpretation of data. Novi Putri Ayu: 

Substantial contributions to acquisition of 

data. Zahra Nurussofa: Substantial 

contributions to conception and design 

Dewi Jantika Djuarna: Substantial 

contributions to conception and design 

Rini Latifah: Substantial contributions to 

conception and design. Wawang Setiawan 

Sukarya: Final approval of the version to be 

published 

 

ACKNOWLEDGMENTS 

The authors would like to thank all 

supporting staff at the Faculty of Medicine 

and thank Lemlit UHAMKA. 

 

CONFLICT OF INTEREST 

The authors declare that there is no 

conflict of interest.

 

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